Mark P. Burns

Cdk5 Is a Key Factor in Tau Aggregation and Tangle Formation In Vivo

Tau aggregation is a common feature of neurodegenerative diseases such as Alzheimers disease, and hyperphosphorylation of tau has been implicated as a fundamental pathogenic mechanism in this process. To examine the impact of cdk5 in tau aggregation and tangle formation, we crossed transgenic mice overexpressing the cdk5 activator p25, with transgenic mice overexpressing mutant (P301L) human tau. Tau was hyperphosphorylated at several sites in the double transgenics, and there was a highly significant accumulation of aggregated tau in brainstem and cortex. This was accompanied by increased numbers of silver-stained neurofibrillary tangles (NFTs). Insoluble tau was also associated with active GSK. Thus, cdk5 can initiate a major impact on tau pathology progression that probably involves several kinases. Kinase inhibitors may thus be beneficial therapeutically.

Amyloid precursor protein secretases as therapeutic targets for traumatic brain injury

Amyloid-β (Aβ) peptides, found in Alzheimers disease brain, accumulate rapidly after traumatic brain injury (TBI) in both humans and animals. Here we show that blocking either β- or γ-secretase, enzymes required for production of Aβ from amyloid precursor protein (APP), can ameliorate motor and cognitive deficits and reduce cell loss after experimental TBI in mice. Thus, APP secretases are promising targets for treatment of TBI.

Traumatic brain injury in aged animals increases lesion size and chronically alters microglial/macrophage classical and alternative activation states

Traumatic brain injury (TBI) causes chronic microglial activation that contributes to subsequent neurodegeneration, with clinical outcomes declining as a function of aging. Microglia/macrophages (MG/Mɸ) have multiple phenotypes, including a classically activated, proinflammatory (M1) state that might contribute to neurotoxicity, and an alternatively activated (M2) state that might promote repair. In this study we used gene expression, immunohistochemical, and stereological analyses to show that TBI in aged versus young mice caused larger lesions associated with an M1/M2 balance switch and increased numbers of reactive (bushy and hypertrophic) MG/Mɸ in the cortex, hippocampus, and thalamus. Chitinase3-like 3 (Ym1), an M2 phenotype marker, displayed heterogeneous expression after TBI with amoeboid-like Ym1-positive MG/Mɸ at the contusion site and ramified Ym1-positive MG/Mɸ at distant sites; this distribution was age-related. Aged-injured mice also showed increased MG/Mɸ expression of major histocompatibility complex II and NADPH oxidase, and reduced antioxidant enzyme expression which was associated with lesion size and neurodegeneration. Thus, altered relative M1/M2 activation and an nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-mediated shift in redox state might contribute to worse outcomes observed in older TBI animals by creating a more proinflammatory M1 MG/Mɸ activation state.

The Effect of Injury Severity on Behavior: A Phenotypic Study of Cognitive and Emotional Deficits after Mild, Moderate, and Severe Controlled Cortical Impact Injury in Mice

Traumatic brain injury (TBI) can cause a broad array of behavioral problems including cognitive and emotional deficits. Human studies comparing neurobehavioral outcomes after TBI suggest that cognitive impairments increase with injury severity, but emotional problems such as anxiety and depression do not. To determine whether cognitive and emotional impairments increase as a function of injury severity we exposed mice to sham, mild, moderate, or severe controlled cortical impact (CCI) and evaluated performance on a variety of neurobehavioral tests in the same animals before assessing lesion volume as a histological measure of injury severity. Increasing cortical impact depth successfully produced lesions of increasing severity in our model. We found that cognitive impairments in the Morris water maze increased with injury severity, as did the degree of contralateral torso flexion, a measure of unilateral striatal damage. TBI also caused deficits in emotional behavior as quantified in the forced swim test, elevated-plus maze, and prepulse inhibition of acoustic startle, but these deficits were not dependent on injury severity. Stepwise regression analyses revealed that Morris water maze performance and torso flexion predicted the majority of the variability in lesion volume. In summary, we find that cognitive deficits increase in relation to injury severity, but emotional deficits do not. Our data suggest that the threshold for emotional changes after experimental TBI is low, with no variation in behavioral deficits seen between mild and severe brain injury.

Cholesterol in Alzheimer's Disease and Tauopathy

Abstract: Cholesterol has been implicated in the pathogenesis of amyloid plaques in Alzheimers disease (AD) and in the formation of neurofibrillary pathology in Niemann‐Pick disease. Several epidemiology studies have implicated high cholesterol as a risk factor for AD and have shown that the use of cholesterol‐reducing agents (statins) can be protective against the disease. We and others have shown that cholesterol levels modulate the processing of the amyloid precursor protein (APP) both in vivo and in vitro, affecting the accumulation of A‐beta (Aβ) peptides that may directly impact the risk of AD. Mutations in the Niemann‐Pick C gene (NPC) result in deficient cholesterol transport/storage. Clinically, Niemann‐Pick disease causes a severe childhood lipidosis, with neurodegeneration characterized by the presence of AD‐type neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau. Studies of mouse models of NPC show that defects in cellular cholesterol trafficking are associated with enhanced generation of Aβ and the hyperphosphorylation of tau, further implicating the cholesterol homeostasis pathway as a risk factor for amyloidosis.

Co-localization of cholesterol, apolipoprotein E and fibrillar Aβ in amyloid plaques

Recent evidence strongly suggests a role for cholesterol and apolipoprotein E in the etiology of Alzheimers disease. We have demonstrated the co-localization of cholesterol and apolipoprotein E with beta-amyloid immunoreactivity and thioflavin S immunofluorescence in AD type plaques of a transgenic mouse model. Cholesterol and apolipoprotein E co-localized to the core of thioflavin S-positive (fibrillar) plaques, but not thioflavin S-negative (diffuse) plaques from an early age. By 18 months of age, there was extensive coverage of fibrillar plaques immunopositive for apolipoprotein E and cholesterol oxidase. These findings support evidence that cholesterol and apolipoprotein E are involved in fibrillar plaque formation or maintenance, and suggest that cholesterol may impact amyloid formation extracellularly, as well as through an intracellular effect.

The effects of ABCA1 on cholesterol efflux and Aβ levels in vitro and in vivo

ABCA1 promotes cholesterol efflux from cells and is required for maintaining plasma cholesterol levels. Cholesterol homeostasis is important in the production of β‐amyloid (Aβ), a peptide that is overproduced in Alzheimers disease (AD). Overexpression of ABCA1 can be achieved by stimulating Liver X Receptors (LXR), and changes in Aβ have been reported after LXR stimulation in vitro. To determine whether ABCA1 could alter endogenous Aβ levels, we used two different in vivo systems. We first examined the effects of an LXR agonist (TO‐901317) on wild‐type mice and found an increase in brain ABCA1 and apoE levels, which caused an increase in plasma cholesterol. This was accompanied by a decrease in brain Aβ levels. We then examined endogenous Aβ levels in ABCA1 knockout mice and found that, despite having no ABCA1, lowered brain apoE levels, and lowered plasma cholesterol, there was no change in Aβ levels. To assess these in vivo models in an in vitro system, we designed a model in which cholesterol transport via ABCA1 (or related transporters) was prevented. Switching off cholesterol efflux, even in the presence of TO‐901317, caused no change in Aβ levels. However, when efflux capability was restored, TO‐901317 reduced Aβ levels. These data show that promoting cholesterol efflux is a viable target for Aβ reducing strategies; however, knockout of cholesterol transporters is not sufficient to alter Aβin vitro or in vivo.

The GABA(A) receptor agonist THIP ameliorates specific behavioral deficits in the mouse model of fragile X syndrome.

Hyperactivity, hypersensitivity to auditory stimuli, and exaggerated fear are common behavioral abnormalities observed in individuals with fragile X syndrome (FXS), a neurodevelopmental disorder that is the most common genetic cause of autism. Evidence from studies of the Fmr1 knockout (KO) mouse model of FXS supports the notion that impaired GABAergic transmission in different brain regions such as the amygdala, striatum or cerebral cortex is central to FXS behavioral abnormalities. This suggests that the GABAergic system might be an intriguing target to ameliorate some of the phenotypes in FXS. Our recent work revealed that THIP (gaboxadol), a GABAA receptor agonist, can restore principal neuron excitability deficits in the Fmr1 KO amygdala, suggesting that THIP may also restore some of the key behavioral abnormalities in Fmr1 KO mice. Here, we reveal that THIP significantly attenuated hyperactivity in Fmr1 KO mice, and reduced prepulse inhibition in a volume-dependent manner. In contrast, THIP did not reverse the deficits in cued fear or startle response. Thus, this study shows that enhancing GABAergic transmission can correct specific behavioral phenotypes of the Fmr1 KO mouse further supporting that targeting the GABAergic system, and specifically tonic inhibition, might be important for correcting or ameliorating some key behaviors in FXS.

Parkin promotes intracellular Aβ1–42 clearance

Alzheimers disease and Parkinsons disease are common neurodegenerative diseases that may share some underlying mechanisms of pathogenesis. Abeta(1-42) fragments are found intracellularly, and extracellularly as amyloid plaques, in Alzheimers disease and in dementia with Lewy Bodies. Parkin is an E3-ubiquitin ligase involved in proteasomal degradation of intracellular proteins. Mutations in parkin, which result in loss of parkin function, lead to early onset Parkinsonism. Here we tested whether the ubiquitin ligase activity of parkin could lead to reduction in intracellular human Abeta(1-42). Lentiviral constructs encoding either human parkin or human Abeta(1-42) were used to infect M17 neuroblastoma cells. Parkin expression resulted in reduction of intracellular human Abeta(1-42) levels and protected against its toxicity in M17 cells. Co-injection of lentiviral constructs into control rat primary motor cortex demonstrated that parkin co-expression reduced human Abeta(1-42) levels and Abeta(1-42)-induced neuronal degeneration in vivo. Parkin increased proteasomal activity, and proteasomal inhibition blocked the effects of parkin on reducing Abeta(1-42) levels. Incubation of Abeta(1-42) cell lysates with ubiquitin, in the presence of parkin, demonstrated the generation of Abeta-ubiquitin complexes. These data indicate that parkin promotes ubiquitination and proteasomal degradation of intracellular Abeta(1-42) and demonstrate a protective effect in neurodegenerative diseases with Abeta deposits.

Low Density Lipoprotein Receptors Regulate Microglial Inflammation Through C-Jun N-Terminal Kinase

Apolipoprotein E (apoE) has been implicated in modulating the central nervous system (CNS) inflammatory response. However, the molecular mechanisms involved in apoE‐dependent immunomodulation are poorly understood. We hypothesize that apoE alters the CNS inflammatory response by signaling via low‐density lipoprotein (LDL) receptors in glia. To address this hypothesis, we used a small bioactive peptide formed from the receptor‐binding domain of apoE, apoE peptide (EP), to study LDL receptor signaling in microglia. To model glial activation, we treated primary mouse microglia and the microglial cell line BV2 with lipopolysaccharide (LPS) and studied two inflammatory responses: an increase in nitric oxide production (NO) and a decrease in apoE production. We found that treatment of primary microglia and BV2 cells with EP attenuated LPS‐induced NO accumulation and apoE reduction in a dose‐dependent manner. Using the receptor‐associated protein to block ligand binding to members of the LDL receptor family, we found that EP attenuated both of these LPS‐induced inflammatory responses via LDL receptors. We studied two intracellular signaling cascades associated with apoE: c‐Jun N‐terminal kinase (JNK) and extracellular signal‐regulated kinase (ERK). LPS induced both ERK and JNK activation, whereas EP induced ERK activation, but drastically reduced JNK activation. Inhibition of JNK with SP600125 reduced LPS‐induced NO production and apoE reduction in a dose‐dependent manner. Treatment of microglia with suboptimal EP in combination with JNK inhibitor enhanced attenuation of LPS‐induced NO production. These data suggest that microglial LDL receptors regulate JNK activation, which is necessary for apoE modulation of the inflammatory response.