Wai Haung Yu

Autophagy Induction and Autophagosome Clearance in Neurons: Relationship to Autophagic Pathology in Alzheimer's Disease

Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the neurodegeneration of Alzheimers disease (AD). The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain, however, is unknown. In this study, we show that constitutive macroautophagy in primary cortical neurons is highly efficient, because newly formed autophagosomes are rapidly cleared by fusion with lysosomes, accounting for their scarcity in the healthy brain. Even after macroautophagy is strongly induced by suppressing mTOR (mammalian target of rapamycin) kinase activity with rapamycin or nutrient deprivation, active cathepsin-positive autolysosomes rather than LC3-II-positive autophagosomes predominate, implying efficient autophagosome clearance in healthy neurons. In contrast, selectively impeding late steps in macroautophagy by inhibiting cathepsin-mediated proteolysis within autolysosomes with cysteine- and aspartyl-protease inhibitors caused a marked accumulation of electron-dense double-membrane-limited AVs, containing cathepsin D and incompletely degraded LC3-II in perikarya and neurites. Similar structures accumulated in large numbers when fusion of autophagosomes with lysosomes was slowed by disrupting their transport on microtubules with vinblastine. Finally, we find that the autophagic vacuoles accumulating after protease inhibition or prolonged vinblastine treatment strongly resembled AVs that collect in dystrophic neurites in the AD brain and in an AD mouse model. We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone. Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway.

Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling

The ubiquitin proteasome system (UPS) degrades misfolded proteins including those implicated in neurodegenerative diseases. We investigated the effects of tau accumulation on proteasome function in a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau was associated with a decrease in the peptidase activity of brain 26S proteasomes, higher levels of ubiquitinated proteins and undegraded Ub-G76V-GFP. 26S proteasomes from mice with tauopathy were physically associated with tau and were less active in hydrolyzing ubiquitinated proteins, small peptides and ATP. 26S proteasomes from normal mice incubated with recombinant oligomers or fibrils also showed lower hydrolyzing capacity in the same assays, implicating tau as a proteotoxin. Administration of an agent that activates cAMP–protein kinase A (PKA) signaling led to attenuation of proteasome dysfunction, probably through proteasome subunit phosphorylation. In vivo, this led to lower levels of aggregated tau and improvements in cognitive performance.

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.

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.

Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory

Non-fibrillar soluble oligomeric forms of amyloid-β peptide (oAβ) and tau proteins are likely to play a major role in Alzheimer’s disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAβ initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aβ, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAβ levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAβ to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aβ on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aβ and tau pathology.