Is Alzheimer disease (AD) a Type 3 diabetes? Previous FDG-PET studies showed that subjects with mild cognitive impairment (MCI) or early AD exhibited reduced glucose utilization and potentially insulin resistance in the brain. A preliminary clinical trial of intranasal insulin therapy showed improved cognitive function in patients with amnestic MCI or AD (Craft et al., 2012). These and other findings provide evidence that glucose utilization and/or insulin receptor function may be disrupted in AD. However, the mechanism(s) underlying this defect remain unclear. Previous studies from our laboratory have shown that the microtubule-related motor protein Eg5/Kinesin 5 is inhibited by Aβ42 oligomers, resulting in chromosome mis-segregation and neurotransmitter and neurotrophin receptor mislocalization and dysfunction (Ari et al., 2010). Such disruption of microtubule-based transport may explain the previous finding of the mis-localization of some, but not all, membrane proteins, such as the insulin receptor (Zhao et al., 2008). In this study, we used immunohistochemistry to examine the effects of treatment with Aβ42 (2 μM) or with the Eg5 inhibitor monastrol (10 μM) on the distribution of insulin receptors and glucose transporters in cultured cells and the localization of the receptors in hippocampus and cortex in a rat model of AD. The results show that glucose transporter distribution in primary rat astrocytes is disrupted by Aβ42 oligomers and by monastrol, resulting in aggregated/clustered glucose transporters in a peri-nuclear region. Insulin receptors also appeared to be clustered in a peri-nuclear region following treatment with Aβ42 (2 μM) or monastrol (10 μM) for 48 h in cultured N2A or PC12 cells. We observed the same results in rat primary cortical neurons. We also found that disruption of insulin receptor distribution could be rescued by Eg5 overexpression in N2A cells. Furthermore, in a rat model of AD, the insulin receptor was abnormally clustered in peri-nuclear regions in the hippocampus and cortex. These findings provide evidence that Aβ42-induced dysregulation of the microtubule network disrupts insulin receptor and glucose transporter trafficking, which is likely to negatively impact neuronal energy metabolism and synaptic plasticity, and suggest that Aβ-mediated inhibition of Eg5/Kinesin 5 may be an important AD therapeutic target.

Microtubule motors are essential for multiple cellular functions, including mitosis and intracellular trafficking of macromolecules and organelles. We have found that oligomeric A b specifically binds to and inhibits several such motors, including kinesin 5/Eg5. As a result, A b leads to defective mitosis, including abnormal spindle structure and chromosome mis-segregation, and may inhibit transport of key cell proteins to the cell surface. We used immunocytochemistry and confocal microscopy to quantitate the localization of cell surface receptors in various cells, including primary neurons. A b treatment reduces transport of the NMDA receptor, and the p75/TrkA neurotrophin receptor to the plasma membrane of neurons and other cells and strongly inhibits their responses their environment. Chemical or A b inhibition of Kinesin 5 reduces neurite outgrowth in response to NGF, Ca++ uptake in response to glutamate in primary neurons and H4 cells and inhibits LTP, a tissue model of learning and memory. APP/A b also inhibits LDLR expression and intracellular localization in N2A cells: the LDL receptor becomes concentrated in a single perinuclear aggregate, rather than sorting to the cell surface. Only light or no LDL staining, indicates that the disruption of receptor distribution also affects its function. We have recently found that this A b -induced defect in MT-based transport extends to the insulin receptor and glucose transporter. For example, the insulin receptor clustered in a perinuclear location when treated with A b for 48 hours in PC 12 cell line. In astrocytes, the glucose transporter distribution is similarly affected by A b treatment, forming an aggregated/cluster near the nucleus, compared to the control. This suggests that energy utilization is altered or less efficient in both astrocytes and neurons exposed to A b, as they are in AD. Overall, these data show disrupted localization and function of essential cell surface proteins in cells exposed to Ab and provide an explanation for the reduced cellular energy usage, smaller neuronal complexity and number, and reduced synaptic plasticity that characterize AD and lead to neurodegeneration and dementia. Similar mitotic and intracellular transport defects characterize many neurodegenerative disease in addition to AD and may represent a common target for therapeutic intervention.

Rheumatoid arthritis (RA) is a negative risk factor for the development of Alzheimer's disease (AD). While it has been commonly assumed that RA patients' usage of non-steroidal anti-inflammatory drugs (NSAIDs) helped prevent onset and progression of AD, NSAID clinical trials have proven unsuccessful in AD patients. To determine whether intrinsic factors within RA pathogenesis itself may underlie RA's protective effect, we investigated the activity of colony-stimulating factors, upregulated in RA, on the pathology and behavior of transgenic AD mice. 5 microg bolus injections of macrophage, granulocyte, and granulocyte-macrophage colony-stimulating factors (M-CSF, G-CSF, or GM-CSF) were administered unilaterally into the hippocampus of aged cognitively-impaired AD mice and the resulting amyloid load reductions determined one week later, using the artificial cerebrospinal fluid-injected contralateral sides as controls. G-CSF and more significantly, GM-CSF reduced amyloidosis throughout the treated brain hemisphere one week following bolus administration to AD mice. 20 daily subcutaneous injections of 5 microg of GM-CSF (the most amyloid-reducing CSF in the bolus experiment) were administered to balanced cohorts of AD mice after assessment in a battery of cognitive tests. Reductions in amyloid load and improvements in cognitive function were assessed. Subcutaneous GM-CSF administration significantly reduced brain amyloidosis and completely reversed the cognitive impairment, while increasing hippocampal synaptic area and microglial density. These findings, along with two decades of accrued safety data using Leukine, recombinant human GMCSF, in elderly leukopenic patients, suggest that Leukine should be tested as a treatment to reverse cerebral amyloid pathology and cognitive impairment in AD.

A common problem faced by researchers using transgenic models to study disease is the phenotypic variability that exists within a group or colony of animals. Significant pathological analyses thus often require large numbers of mice to perform. Many lines of transgenic mice harboring the gene for human amyloid precursor protein (APP) with different mutations causing familial Alzheimer's disease have been developed over the past decade to study plaque deposition and other aspects of AD. However, variations in size, density, plaque number, and total amyloid load between animals of the same age and genotype have been identified by our lab and others. Therefore, to study the effects of compounds on amyloid pathology, it was imperative to develop a technique that would allow each brain hemisphere to receive different infusions. We have developed catheters that facilitate simultaneous bilateral infusion in mouse brains, thereby using the contralateral hemisphere of the same animal as an internal control while studying, for example, the effect of compounds on amyloid plaques, a pathological hallmark of the progression of Alzheimer's disease (AD). Several molecules have been identified within the plaques including the major component, the Aβ peptide, and two inflammation-related proteins, apolipoprotein E (apoE) and the serine protease inhibitor α-1-antichymotrypsin (ACT). In these experiments, ACT was infused unilaterally over a period of 28 days into the parenchyma and lateral ventricles of PS/APP mice and observed to associate with amyloid plaques, with minimal mortality. Utilizing the ACT/Aβ interaction, details of this procedure are discussed here in detail.

In addition to neuritic plaques and neurofibrillary tangles, Alzheimer's disease is characterized by neuroinflammation and neuronal loss. The association of proteins such as apolipoprotein E, alpha 1-antichymotrypsin (ACT), complement factors, and cytokines with the plaques led to the hypothesis that these inflammatory proteins play a role in the development of AD pathology. Studies from our lab and others have shown that ACT induces accelerated plaque formation and cognitive deficit in APP transgenic mice. Recently we showed that ACT also induces tau hyperphorylation and these tau epitopes are similar to the ones that are associated with neurofibrillary tangles. In vitro studies with ACT were done in cultured cortical neurons. Tau phosphorylation in cells and sections were determined by immunohistochemistry. In vivo studies were done in mice expressing APP, human tau or ACT transgenes. ACT infusion was done using alzet pump. To determine whether the effect of ACT on tau hyperphosphorylation was A-beta dependent, we performed experiments in cultured neurons from APP knockout mice. We found that ACT induces tau hyperphosphorylation in these neurons suggesting that the effect of ACT is not A-beta dependent. To study the in vivo effect of ACT, it was infused into the hippocampus on one hemisphere of the mouse brain while the other hemisphere received artificial CSF for the same period of time. After two weeks of infusion the mice were sacrificed and tau phosphorylation determined using specific P-tau antibodies. We found that the hemisphere that received ACT showed an induction of tau hyperphosphorylation compared to the hemisphere that received artificial CSF. This further confirms that inflammatory proteins, namely ACT induces tangle pathology. To further understand the mechanisms involved in ACT's effect on tau, transgenic mice overexpressing human tau in a mouse tau null background (htau) were crossed with human ACT expressing mice. Preliminary studies suggest that mice expressing htau/ACT show increased tau hyperphosphorylation compared to age-matched htau only mice. ACT induces tau hyperphosphorylation in vitro and in vivo independent of A-beta. htau/ACT mice are excellent models for studying age-dependent changes in inflammation-induced neurodegeneration as well as assessing the efficacy of anti-inflammatory drugs.

BackgroundThe blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.Methodology/Principal FindingsEvans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.Conclusions/SignificanceResults suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.