Helen Y. Figueroa

Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons

Background: Exogenous, misfolded Tau can be internalized, but details of the mechanism are unknown. Results: Small misfolded Tau species are internalized through endocytosis, anterogradely and retrogradely transported. Conclusion: Tau uptake is dependent on conformation and size of aggregates, and regulated through endocytosis. Significance: Understanding the mechanism by which pathological Tau is internalized provides a foundation for therapeutic approaches targeting uptake and propagation of tauopathy. The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845–12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909–913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

Transcriptional Regulation of β-Secretase by p25/cdk5 Leads to Enhanced Amyloidogenic Processing

Cyclin-dependent kinase 5 (cdk5) has been implicated in Alzheimers disease (AD) pathogenesis. Here, we demonstrate that overexpression of p25, an activator of cdk5, led to increased levels of BACE1 mRNA and protein in vitro and in vivo. A p25/cdk5 responsive region containing multiple sites for signal transducer and activator of transcription (STAT1/3) was identified in the BACE1 promoter. STAT3 interacts with the BACE1 promoter, and p25-overexpressing mice had elevated levels of pSTAT3 and BACE1, whereas cdk5-deficient mice had reduced levels. Furthermore, mice with a targeted mutation in the STAT3 cdk5 responsive site had lower levels of BACE1. Increased BACE levels in p25 overexpressing mice correlated with enhanced amyloidogenic processing that could be reversed by a cdk5 inhibitor. These data demonstrate a pathway by which p25/cdk5 increases the amyloidogenic processing of APP through STAT3-mediated transcriptional control of BACE1 that could have implications for AD pathogenesis.

Interplay between Cyclin-Dependent Kinase 5 and Glycogen Synthase Kinase 3β Mediated by Neuregulin Signaling Leads to Differential Effects on Tau Phosphorylation and Amyloid Precursor Protein Processing

Cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase 3β (GSK3β) have been implicated in pathogenic processes associated with Alzheimers disease because both kinases regulate tau hyperphosphorylation and enhance amyloid precursor protein (APP) processing leading to an increase in amyloid β (Aβ) production. Here we show that young p25 overexpressing mice have enhanced cdk5 activity but reduced GSK3β activity attributable to phosphorylation at the inhibitory GSK3β–serine 9 (GSK3β–S9) site. Phosphorylation at this site was mediated by enhanced activity of the neuregulin receptor complex, ErbB, and activation of the downstream phosphatidylinositol 3 kinase/Akt pathway. Young p25 mice had elevated Aβ peptide levels, but phospho-tau levels were decreased overall. Thus, cdk5 appears to play a dominant role in the regulation of amyloidogenic APP processing, whereas GSK3β plays a dominant role in overall tau phosphorylation. In older mice, GSK3β inhibitory phosphorylation at S9 was reduced relative to young mice. Aβ peptide levels were still elevated but phospho-tau levels were either unchanged or showed a trend to increase, suggesting that GSK3β activity increases with aging. Inhibition of cdk5 by a specific inhibitor reduced cdk5 activity in p25 mice, leading to reduced Aβ production in both young and old mice. However, in young mice, cdk5 inhibition reversed GSK3β inhibition, leading to an increase in overall tau phosphorylation. When cdk5 inhibitor was administered to very old, nontransgenic mice, inhibition of cdk5 reduced Aβ levels, and phospho-tau levels showed a trend to increase. Thus, cdk5 inhibitors may not be effective in targeting tau phosphorylation in the elderly.

Metabolic Activity Determines Efficacy of Macroautophagic Clearance of Pathological Oligomeric α-Synuclein

Macroautophagy is an essential degradative pathway that can be induced to clear aggregated proteins, such as those found in Parkinsons disease and dementia with Lewy bodies, a form of Parkinsonism. This study found that both LC3-II and beclin were significantly increased in brains from humans with Dementia with Lewy bodies and transgenic mice overexpressing mutant alpha-synuclein, as compared with respective controls, suggesting that macroautophagy is induced to remove alpha-syn, particularly oligomeric or mutant forms. Aged mutant animals had higher autophagy biomarker levels relative to younger animals, suggesting that with aging, autophagy is less efficient and requires more stimulation to achieve the same outcome. Disruption of autophagy by RNA interference significantly increased alpha-syn oligomer accumulation in vitro, confirming the significance of autophagy in alpha-syn clearance. Finally, rotenone-induced alpha-syn aggregates were cleared following rapamycin stimulation of autophagy. Chronic rotenone exposure and commensurate reduction of metabolic activity limited the efficacy of rapamycin to promote autophagy, suggesting that cellular metabolism is critical for determining autophagic activity. Cumulatively, these findings support the concept that neuronal autophagy is essential for protein homeostasis and, in our system, reduction of autophagy increased the accumulation of potentially pathogenic alpha-synuclein oligomers. Aging and metabolic state were identified as important determinants of autophagic activity. This study provides therapeutic and pathological implications for both synucleinopathy and Parkinsons disease, identifying conditions in which autophagy may be insufficient to degrade alpha-syn aggregates.

A transgenic rat that develops Alzheimer's disease-like amyloid pathology, deficits in synaptic plasticity and cognitive impairment

In the last decade, multiple lines of transgenic APP overexpressing mice have been created that recapitulate certain aspects of Alzheimers disease (AD). However, none of the previously reported transgenic APP overexpressing rat models developed AD-like beta-amyloid (Abeta) deposits, or age-related learning and memory deficits. In the present study, we have characterized a transgenic rat model overexpressing transgenes with three, familial AD mutations (two in APP and one in PS1) that were developed by Flood et al. [Flood, D.G., et al., Abeta deposition in a transgenic rat model of Alzheimers disease. Society for Neuroscience 2003, Washington, DC, 2003]. From the age of 9 months, these rats develop Abeta deposits in both diffuse and compact forms, with the latter being closely associated with activated microglia and reactive astrocytes. Impaired long-term potentiation (LTP) was revealed by electrophysiological recordings performed on hippocampal slices from rats at 7 months of age, which is 2 months before the appearance of amyloid plaques. The deficit in LTP was accompanied by impaired spatial learning and memory in the Morris water maze, which became more pronounced in transgenic rats of 13 months of age. For Tg rats of both ages, there was a trend for cognitive impairment to correlate with total Abeta42 levels in the hippocampus. The rat model therefore recapitulates AD-like amyloid pathology and cognitive impairment. The advantage of the rat model over the available mouse models is that rats provide better opportunities for advanced studies, such as serial CSF sampling, electrophysiology, neuroimaging, cell-based transplant manipulations, and complex behavioral testing.

Anesthesia-Induced Hyperphosphorylation Detaches 3-Repeat Tau from Microtubules without Affecting Their Stability In Vivo

In Alzheimers disease, tau is hyperphosphorylated, which is thought to detach it from microtubules (MTs), induce MT destabilization, and promote aggregation. Using a previously described in vivo model, we investigated whether hyperphosphorylation impacts tau function in wild-type and transgenic mice. We found that after anesthesia-induced hypothermia, MT-free tau was hyperphosphorylated, which impaired its ability to bind MTs and promote MT assembly. MT-bound tau was more resistant to hyperphosphorylation compared with free tau and tau did not dissociate from MTs in wild-type mice. However, 3-repeat tau detached from MT in the transgenic mice. Surprisingly, dissociation of tau from MTs did not lead to overt depolymerization of tubulin, and there was no collapse, or disturbance of axonal MT networks. These results indicate that, in vivo, a subpopulation of tau bound to MTs does not easily dissociate under conditions that extensively phosphorylate tau. Tau remaining on the MTs under these conditions is sufficient to maintain MT network integrity.

Tau Pathology Induces Excitatory Neuron Loss, Grid Cell Dysfunction, and Spatial Memory Deficits Reminiscent of Early Alzheimer’s Disease

The earliest stages of Alzheimers disease (AD) are characterized by the formation of mature tangles in the entorhinal cortex and disorientation and confusion when navigating familiar places. The medial entorhinal cortex (MEC) contains specialized neurons called grid cells that form part of the spatial navigation system. Here we show in a transgenic mouse model expressing mutant human tau predominantly in the EC that the formation of mature tangles in old mice was associated with excitatory cell loss and deficits in grid cell function, including destabilized grid fields and reduced firing rates, as well as altered network activity. Overt tau pathology in the aged mice was accompanied by spatial memory deficits. Therefore, tau pathology initiated in the entorhinal cortex could lead to deficits in grid cell firing and underlie the deterioration of spatial cognition seen in human AD.

Neuronal hyperactivity due to loss of inhibitory tone in APOE4 mice lacking Alzheimer’s disease-like pathology

The ε4 allele of apolipoprotein E (APOE) is the dominant genetic risk factor for late-onset Alzheimer’s disease (AD). However, the reason APOE4 is associated with increased AD risk remains a source of debate. Neuronal hyperactivity is an early phenotype in both AD mouse models and in human AD, which may play a direct role in the pathogenesis of the disease. Here, we have identified an APOE4-associated hyperactivity phenotype in the brains of aged APOE mice using four complimentary techniques—fMRI, in vitro electrophysiology, in vivo electrophysiology, and metabolomics—with the most prominent hyperactivity occurring in the entorhinal cortex. Further analysis revealed that this neuronal hyperactivity is driven by decreased background inhibition caused by reduced responsiveness of excitatory neurons to GABAergic inhibitory inputs. Given the observations of neuronal hyperactivity in prodromal AD, we propose that this APOE4-driven hyperactivity may be a causative factor driving increased risk of AD among APOE4 carriers.The APOE4 allele is the leading risk factor for late-onset Alzheimer’s disease, but how it might contribute to the disease is not clear. Here the authors show that a mouse expressing the human APOE4 allele displays hyperactivity in the entorhinal cortex due to a decreased inhibitory tone, which may in part explain accelerated Alzheimer’s pathology in APOE4 carriers.

3D Visualization of the Temporal and Spatial Spread of Tau Pathology Reveals Extensive Sites of Tau Accumulation Associated with Neuronal Loss and Recognition Memory Deficit in Aged Tau Transgenic Mice.

3D volume imaging using iDISCO+ was applied to observe the spatial and temporal progression of tau pathology in deep structures of the brain of a mouse model that recapitulates the earliest stages of Alzheimer’s disease (AD). Tau pathology was compared at four timepoints, up to 34 months as it spread through the hippocampal formation and out into the neocortex along an anatomically connected route. Tau pathology was associated with significant gliosis. No evidence for uptake and accumulation of tau by glia was observed. Neuronal cells did appear to have internalized tau, including in extrahippocampal areas as a small proportion of cells that had accumulated human tau protein did not express detectible levels of human tau mRNA. At the oldest timepoint, mature tau pathology in the entorhinal cortex (EC) was associated with significant cell loss. As in human AD, mature tau pathology in the EC and the presence of tau pathology in the neocortex correlated with cognitive impairment. 3D volume imaging is an ideal technique to easily monitor the spread of pathology over time in models of disease progression.

The Endosomal–Lysosomal Pathway Is Dysregulated by APOE4 Expression in Vivo

Possession of the ε4 allele of apolipoprotein E (APOE) is the major genetic risk factor for late-onset Alzheimers disease (AD). Although numerous hypotheses have been proposed, the precise cause of this increased AD risk is not yet known. In order to gain a more comprehensive understanding of APOE4s role in AD, we performed RNA-sequencing on an AD-vulnerable vs. an AD-resistant brain region from aged APOE targeted replacement mice. This transcriptomics analysis revealed a significant enrichment of genes involved in endosomal–lysosomal processing, suggesting an APOE4-specific endosomal–lysosomal pathway dysregulation in the brains of APOE4 mice. Further analysis revealed clear differences in the morphology of endosomal–lysosomal compartments, including an age-dependent increase in the number and size of early endosomes in APOE4 mice. These findings directly link the APOE4 genotype to endosomal–lysosomal dysregulation in an in vivo, AD pathology-free setting, which may play a causative role in the increased incidence of AD among APOE4 carriers.