William J. Netzer

Chaperones increase association of tau protein with microtubules

Molecular chaperones and their functions in protein folding have been implicated in several neurodegenerative diseases, including Parkinsons disease and Huntingtons disease, which are characterized by accumulation of protein aggregates (e.g., α-synuclein and huntingtin, respectively). These aggregates have been shown in various experimental systems to respond to changes in levels of molecular chaperones suggesting the possibility of therapeutic intervention and a role for chaperones in disease pathogenesis. It remains unclear whether chaperones also play a role in Alzheimers disease, a neurodegenerative disorder characterized by β-amyloid and tau protein aggregates. Here, we report an inverse relationship between aggregated tau and the levels of heat shock protein (Hsp)70/90 in tau transgenic mouse and Alzheimers disease brains. In various cellular models, increased levels of Hsp70 and Hsp90 promote tau solubility and tau binding to microtubules, reduce insoluble tau and cause reduced tau phosphorylation. Conversely, lowered levels of Hsp70 and Hsp90 result in the opposite effects. We have also demonstrated a direct association of the chaperones with tau proteins. Our results suggest that up-regulation of molecular chaperones may suppress formation of neurofibrillary tangles by partitioning tau into a productive folding pathway and thereby preventing tau aggregation.

Inhibition of amyloid-β aggregation and caspase-3 activation by the Ginkgo biloba extract EGb761

Standardized extract from the leaves of the Ginkgo biloba tree, labeled EGb761, has been used in clinical trials for its beneficial effects on brain functions, particularly in connection with age-related dementias and Alzheimers disease (AD). Substantial experimental evidence indicates that EGb761 protects against neuronal damage from a variety of insults, but its cellular and molecular mechanisms remain unknown. Using a neuroblastoma cell line stably expressing an AD-associated double mutation, we report that EGb761 inhibits formation of amyloid-β (Aβ) fibrils, which are the diagnostic, and possibly causative, feature of AD. The decreased Aβ fibrillogenesis in the presence of EGb761 was observed both in the conditioned medium of this Aβ-secreting cell line and in solution in vitro. In the cells, EGb761 significantly attenuated mitochondrion-initiated apoptosis and decreased the activity of caspase 3, a key enzyme in the apoptosis cell-signaling cascade. These results suggest that (i) neuronal damage in AD might be due to two factors: a direct Aβ toxicity and the apoptosis initiated by the mitochondria; and (ii) multiple cellular and molecular neuroprotective mechanisms, including attenuation of apoptosis and direct inhibition of Aβ aggregation, underlie the neuroprotective effects of EGb761.

Does insulin dysfunction play a role in Alzheimer's disease?

Age-related changes in hormone levels are determinants of a variety of human diseases. Insulin is known to affect numerous brain functions including cognition and memory, and several clinical studies have established links between Alzheimers disease (AD), insulin resistance and diabetes mellitus. These are reinforced by biological studies that reveal the effects of insulin on the molecular and cellular mechanisms that underlie the pathology of AD. For example, insulin regulates phosphorylation of tau protein, which underlies neurofibrillary lesions in the brains of AD patients. Insulin also affects the metabolism of beta-amyloid, the main constituent of AD amyloid pathology. Here, we discuss clinical and biological data that highlight potential targets for therapeutic intervention.

Gamma-secretase activating protein is a therapeutic target for Alzheimer’s disease

Accumulation of neurotoxic amyloid-β is a major hallmark of Alzheimer’s disease. Formation of amyloid-β is catalysed by γ-secretase, a protease with numerous substrates. Little is known about the molecular mechanisms that confer substrate specificity on this potentially promiscuous enzyme. Knowledge of the mechanisms underlying its selectivity is critical for the development of clinically effective γ-secretase inhibitors that can reduce amyloid-β formation without impairing cleavage of other γ-secretase substrates, especially Notch, which is essential for normal biological functions. Here we report the discovery of a novel γ-secretase activating protein (GSAP) that drastically and selectively increases amyloid-β production through a mechanism involving its interactions with both γ-secretase and its substrate, the amyloid precursor protein carboxy-terminal fragment (APP-CTF). GSAP does not interact with Notch, nor does it affect its cleavage. Recombinant GSAP stimulates amyloid-β production in vitro. Reducing GSAP concentrations in cell lines decreases amyloid-β concentrations. Knockdown of GSAP in a mouse model of Alzheimer’s disease reduces levels of amyloid-β and plaque development. GSAP represents a type of γ-secretase regulator that directs enzyme specificity by interacting with a specific substrate. We demonstrate that imatinib, an anticancer drug previously found to inhibit amyloid-β formation without affecting Notch cleavage, achieves its amyloid-β-lowering effect by preventing GSAP interaction with the γ-secretase substrate, APP-CTF. Thus, GSAP can serve as an amyloid-β-lowering therapeutic target without affecting other key functions of γ-secretase.

Overactivation of glycogen synthase kinase-3 by inhibition of phosphoinositol-3 kinase and protein kinase C leads to hyperphosphorylation of tau and impairment of spatial memory

Neurofibrillary tangles (NFTs) consisting of the hyperphosphorylated microtubule‐associated protein tau are a defining pathological characteristic of Alzheimers disease (AD). Hyperphosphorylation of tau is hypothesized to impair the microtubule stabilizing function of tau, leading to the formation of paired helical filaments and neuronal death. Glycogen synthase kinase‐3 (GSK‐3) has been shown to be one of several kinases that mediate tau hyperphosphorylation in vitro. However, molecular mechanisms underlying overactivation of GSK‐3 and its potential linkage to AD‐like pathologies in vivo remain unclear. Here, we demonstrate that injection of wortmannin (a specific inhibitor of phosphoinositol‐3 kinase) or GF‐109203X (a specific inhibitor of protein kinase C) into the left ventricle of rat brains leads to overactivation of GSK‐3, hyperphosphorylation of tau at Ser 396/404/199/202 and, most significantly, impaired spatial memory. The effects of wortmannin and GF‐109203X are additive. Significantly, specific inhibition of GSK‐3 activity by LiCl prevents hyperphosphorylation of tau, and spatial memory impairment resulting from PI3K and PKC inhibition. These results indicate that in vivo inhibition of phosphoinositol‐3 kinase and protein kinase C results in overactivation of GSK‐3 and tau hyperphosphorylation and support a direct role of GSK‐3 in the formation of AD‐like cognitive deficits.

Gleevec inhibits β-amyloid production but not Notch cleavage

Amyloid-β (Aβ) peptides, consisting mainly of 40 and 42 aa (Aβ40 and Aβ42, respectively), are metabolites of the amyloid precursor protein and are believed to be major pathological determinants of Alzheimers disease. The proteolytic cleavages that form the Aβ N and C termini are catalyzed by β-secretase and γ-secretase, respectively. Here we demonstrate that γ-secretase generation of Aβ in an N2a cell-free system is ATP dependent. In addition, the Abl kinase inhibitor imatinib mesylate (Gleevec, or STI571), which targets the ATP-binding site of Abl and several other tyrosine kinases, potently reduces Aβ production in the N2a cell-free system and in intact N2a cells. Both STI571 and a related compound, inhibitor 2, also reduce Aβ production in rat primary neuronal cultures and in vivo in guinea pig brain. STI571 does not inhibit the γ-secretase-catalyzed S3 cleavage of Notch-1. Furthermore, production of Aβ and its inhibition by STI571 were demonstrated to occur to similar extents in both Abl-/- and WT mouse fibroblasts, indicating that the effect of STI571 on Aβ production does not involve Abl kinase. The efficacy of STI571 in reducing Aβ without affecting Notch-1 cleavage may prove useful as a basis for developing novel therapies for Alzheimers disease.

β-Amyloid Modulation of Synaptic Transmission and Plasticity

The sequencing of β amyloid protein (Aβ) in 1984 led to the formulation of the “amyloid hypothesis” of Alzheimers disease (AD) ([Glenner and Wong, 1984][1]). The hypothesis proposed that accumulation of Aβ is responsible for AD-related pathology, including Aβ deposits, neurofibrillary

Lowering β-Amyloid Levels Rescues Learning and Memory in a Down Syndrome Mouse Model

β-amyloid levels are elevated in Down syndrome (DS) patients throughout life and are believed to cause Alzheimers disease (AD) in adult members of this population. However, it is not known if β-amyloid contributes to intellectual disability in younger individuals. We used a γ-secretase inhibitor to lower β-amyloid levels in young mice that model DS. This treatment corrected learning deficits characteristic of these mice, suggesting that β-amyloid-lowering therapies might improve cognitive function in young DS patients.

A Functional Mouse Retroposed Gene Rps23r1 Reduces Alzheimer's β-Amyloid Levels and Tau Phosphorylation

Senile plaques consisting of beta-amyloid (Abeta) and neurofibrillary tangles composed of hyperphosphorylated tau are major pathological hallmarks of Alzheimers disease (AD). Elucidation of factors that modulate Abeta generation and tau hyperphosphorylation is crucial for AD intervention. Here, we identify a mouse gene Rps23r1 that originated through retroposition of ribosomal protein S23. We demonstrate that RPS23R1 protein reduces the levels of Abeta and tau phosphorylation by interacting with adenylate cyclases to activate cAMP/PKA and thus inhibit GSK-3 activity. The function of Rps23r1 is demonstrated in cells of various species including human, and in transgenic mice overexpressing RPS23R1. Furthermore, the AD-like pathologies of triple transgenic AD mice were improved and levels of synaptic maker proteins increased after crossing them with Rps23r1 transgenic mice. Our studies reveal a new target/pathway for regulating AD pathologies and uncover a retrogene and its role in regulating protein kinase pathways.

Reduction of Synaptojanin 1 Accelerates Aβ Clearance and Attenuates Cognitive Deterioration in an Alzheimer Mouse Model

Background: Recent studies have linked synaptojanin 1 (synj1) with Alzheimer disease (AD). Results: We report that synj1 reduction decreases amyloid plaque burden and attenuates cognitive deterioration in an AD mouse model. These effects are mediated through accelerating endosomal/lysosomal degradation of Aβ. Conclusion: Our data suggest a novel mechanism by which synj1 reduction promotes Aβ clearance. Significance: These studies implicate a therapeutic strategy for AD. Recent studies link synaptojanin 1 (synj1), the main phosphoinositol (4,5)-biphosphate phosphatase (PI(4,5)P2-degrading enzyme) in the brain and synapses, to Alzheimer disease. Here we report a novel mechanism by which synj1 reversely regulates cellular clearance of amyloid-β (Aβ). Genetic down-regulation of synj1 reduces both extracellular and intracellular Aβ levels in N2a cells stably expressing the Swedish mutant of amyloid precursor protein (APP). Moreover, synj1 haploinsufficiency in an Alzheimer disease transgenic mouse model expressing the Swedish mutant APP and the presenilin-1 mutant ΔE9 reduces amyloid plaque load, as well as Aβ40 and Aβ42 levels in hippocampus of 9-month-old animals. Reduced expression of synj1 attenuates cognitive deficits in these transgenic mice. However, reduction of synj1 does not affect levels of full-length APP and the C-terminal fragment, suggesting that Aβ generation by β- and γ-secretase cleavage is not affected. Instead, synj1 knockdown increases Aβ uptake and cellular degradation through accelerated delivery to lysosomes. These effects are partially dependent upon elevated PI(4,5)P2 with synj1 down-regulation. In summary, our data suggest a novel mechanism by which reduction of a PI(4,5)P2-degrading enzyme, synj1, improves amyloid-induced neuropathology and behavior deficits through accelerating cellular Aβ clearance.