Neurofibrillary tangles composed of aggregated, hyperphosphorylated tau in an abnormal conformation represent one of the major pathological hallmarks of Alzheimer's disease (AD) and other ...tauopathies. However, recent data suggest that the pathogenic processes leading to cognitive impairment occur before the formation of classic tangles. In the earliest stages of tauopathy, tau detaches from microtubules and accumulates in the cytosol of the somatodendritic compartment of cells. Either as a cause or an effect, tau becomes hyperphosphorylated and aggregates into paired helical filaments that comprise the tangles. To assess whether an agent that modulates microtubule function can inhibit the pathogenic process and prevent cognitive deficits in a transgenic mouse model with AD-relevant tau pathology, we administered the neuronal tubulin-preferring agent, NAPVSIPQ (NAP). Three months of treatment with NAP at an early-to-moderate stage of tauopathy reduced the levels of hyperphosphorylated soluble and insoluble tau. A 6-month course of treatment improved cognitive function. Although nonspecific tubulin-interacting agents commonly used for cancer therapy are associated with adverse effects due to their anti-mitotic activity, no adverse effects were found after 6 months of exposure to NAP. Our results suggest that neuronal microtubule interacting agents such as NAP may be useful therapeutic agents for the treatment or prevention of tauopathies.
Neurofibrillary tangles (NFT), mainly consisting of fibrillar aggregates of hyperphosphorylated tau, are a defining pathological feature of Alzheimer's Disease and other tauopathies. Progressive ...accumulation of tau into NFT is considered to be a toxic cellular event causing neurodegeneration. Tau is subject to O-linked N-acetylglucosamine (O-GlcNAc) modification and O-GlcNAcylation of tau has been suggested to regulate tau phosphorylation. We tested if an increase in tau O-GlcNAcylation affected tau phosphorylation and aggregation in the rTg4510 tau transgenic mouse model. Acute treatment of rTg4510 mice with an O-GlcNAcase inhibitor transiently reduced tau phosphorylation at epitopes implicated in tau pathology. More importantly, long-term inhibitor treatment strongly increased tau O-GlcNAcylation, reduced the number of dystrophic neurons, and protected against the formation of pathological tau species without altering the phosphorylation of non-pathological tau. This indicates that O-GlcNAcylation prevents the aggregation of tau in a manner that does not affect its normal phosphorylation state. Collectively, our results support O-GlcNAcase inhibition as a potential therapeutic strategy for the treatment of Alzheimer's Disease and other tauopathies.
•Acute OGA inhibition transiently reduced tau phosphorylation in rTg4510 mice.•Chronic OGA inhibition protected against tau aggregation in rTg4510 mice.•Chronic OGA inhibition did not affect normal tau phosphorylation in rTg4510 mice.
Abstract Epidemiological studies indicate that women have a higher risk of Alzheimer's disease (AD) even after adjustment for age. Though transgenic mouse models of AD develop AD-related amyloid beta ...(Abeta) and/or tau pathology, gender differences have not been well documented in these models. In this study, we found that female 3xTg-AD transgenic mice expressing mutant APP, presenilin-1 and tau have significantly more aggressive Abeta pathology. We also found an increase in beta-secretase activity and a reduction of neprilysin in female mice compared to males; this suggests that a combination of increased Abeta production and decreased Abeta degradation may contribute to higher risk of AD in females. In contrast to significantly more aggressive Abeta pathology in females, gender did not affect the levels of phosphorylated tau in 3xTg-AD mice. These results point to the involvement of Abeta pathways in the higher risk of AD in women. In addition to comparison of pathology between genders at 9, 16 and 23 months of age, we examined the progression of Abeta pathology at additional age points; i.e., brain Abeta load, intraneuronal oligomeric Abeta distribution and plaque load, in male 3xTg-AD mice at 3, 6, 9, 12, 16, 20 and 23 months of age. These findings confirm progressive Abeta pathology in 3xTg-AD transgenic mice, and provide guidance for their use in therapeutic research.
Accumulation of beta-amyloid (Abeta) peptide and hyperphosphorylation of tau in the brain are pathological hallmarks of Alzheimer's disease (AD). Agents altering these pathological events might ...modify clinical disease progression. NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln) is an octapeptide that has shown neuroprotective effects in various in vitro and in vivo neurodegenerative models. Previous studies showed that NAP protected against Abeta-induced neurotoxicity, inhibited Abeta aggregation, and, by binding to tubulin, prevented disruption of microtubules. In this study, we investigated the effect of NAP on Abeta and tau pathology using a transgenic mouse model that recapitulates both aspects of AD. We administered NAP intranasally (0.5 microg/mouse per day, daily from Monday through Friday) for 3 mo, starting from 9 mo of age, which is a prepathological stage in these mice. NAP treatment significantly lowered levels of Abeta 1-40 and 1-42 in brain. In addition, NAP significantly reduced levels of hyperphosphorylated tau. Of particular interest, hyperphosphorylation at the threonine 231 site was reduced; phosphorylation at this site influences microtubule binding. Our results indicate that NAP treatment of transgenic mice initiated at an early stage reduced both Abeta and tau pathology, suggesting that NAP might be a potential therapeutic agent for AD.
A history of depression is a risk factor for Alzheimer's disease (AD), suggesting the possibility that antidepressants administered prophylactically might retard the disease process and preserve ...cognitive function. Here we report that pre-symptomatic treatment with the antidepressant paroxetine attenuates the disease process and improves cognitive performance in the 3xTgAD mouse model of AD. Five-month-old male and female 3xTgAD and non-transgenic mice were administered either paroxetine or saline daily for 5 months. Open-field activity was tested in 7-month-old mice and performance in passive avoidance and Morris swim tasks were evaluated at 10 months. 3xTgAD mice exhibited reduced exploratory activity, increased transfer latency in the passive avoidance test and impaired performance in the Morris spatial navigation task compared to nontransgenic control mice. Paroxetine treatment ameliorated the spatial navigation deficit in 3xTgAD male and female mice, without affecting swim speed or distance traveled, suggesting a preservation of cognitive function. Levels of amyloid beta-peptide (Aβ) and numbers of Aβ immunoreactive neurons were significantly reduced in the hippocampus of male and female paroxetine-treated 3xTgAD mice compared to saline-treated 3xTgAD mice. Female 3xTgAD mice exhibited significantly less tau pathology in the hippocampus and amygdala compared to male 3xTgAD mice, and paroxetine lessened tau pathology in male 3xTgAD mice. The ability of a safe and effective antidepressant to suppress neuropathological changes and improve cognitive performance in a mouse model suggests that such drugs administered prophylactically might retard the development of AD in humans.
Accumulation of amyloid beta peptide (Abeta) in brain is a hallmark of Alzheimer's disease (AD). Inhibition of beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE1), the enzyme that ...initiates Abeta production, and other Abeta-lowering strategies are commonly tested in transgenic mice overexpressing mutant APP. However, sporadic AD cases, which represent the majority of AD patients, are free from the mutation and do not necessarily have overproduction of APP. In addition, the commonly used Swedish mutant APP alters APP cleavage. Therefore, testing Abeta-lowering strategies in transgenic mice may not be optimal. In this study, we investigated the impact of BACE1 inhibition in non-transgenic mice with physiologically relevant APP expression. Existing Abeta ELISAs are either relatively insensitive to mouse Abeta or not specific to full-length Abeta. A newly developed ELISA detected a significant reduction of full-length soluble Abeta 1-40 in mice with the BACE1 homozygous gene deletion or BACE1 inhibitor treatment, while the level of x-40 Abeta was moderately reduced due to detection of non-full-length Abeta and compensatory activation of alpha-secretase. These results confirmed the feasibility of Abeta reduction through BACE1 inhibition under physiological conditions. Studies using our new ELISA in non-transgenic mice provide more accurate evaluation of Abeta-reducing strategies than was previously feasible.
Phospholipase C‐η2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLCη2 activity is triggered by intracellular calcium ...mobilization and likely serves to amplify Ca2+ signals by stimulating further Ca2+ release from Ins(1,4,5)P3‐sensitive stores. The role of PLCη2 in neuritogenesis was assessed during retinoic acid (RA)‐induced Neuro2A cell differentiation. PLCη2 expression increased two‐fold during a 4‐day differentiation period. Stable expression of PLCη2‐targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA‐treatment. Furthermore, RA response element activation was perturbed by PLCη2 knockdown. Using a bacterial two‐hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLCη2. Immunostaining of PLCη2 revealed significant co‐localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA‐induced phosphorylation of LIMK1 and cAMP‐responsive element‐binding protein was reduced in PLCη2 knock‐down cells. The phosphoinositide‐binding properties of the PLCη2 PH domain, assessed using a FRET‐based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P3. Immunostaining of PLCη2 together with PtdIns(3,4,5)P3 in the Neuro2A cells revealed a high degree of co‐localization, indicating that PtdIns(3,4,5)P3 levels in cellular compartments are likely to be important for the spatial control of PLCη2 signaling.
PLCη2 is a Ca2+‐responsive phospholipase C implicated in the regulation of neuronal differentiation/maturation. Targeted knockdown of PLCη2 expression in retinoic acid‐stimulated Neuro2A cells impacted upon neurite growth, RARE activation and LIMK1/CREB signaling. The PLCη2 PH domain was also found to possess a high affinity toward PtdIns(3,4,5)P3, suggesting this phospholipid to be important for the spatial control of PLCη2 signaling.
Alzheimer’s disease (AD) is a neurodegenerative affliction associated with memory dysfunction. Senile plaques are a pathological hallmark of AD, and amyloid β (Aβ) peptides are a major component of ...these plaques. Aβ peptides are derived from proteolytic cleavage of the Aβ protein precursor (APP) by β- and γ-secretases to generate two principal species, Aβ1–40 and Aβ1–42. We have developed antibodies against the N- and C-termini of these peptides, and an ELISA for accurate and sensitive quantitative assessment. Sandwich ELISA composed of N-terminus (Aβ1) end-specific antibody, clone 82E1, and C-termini end-specific antibodies, and clones 1A10 and 1C3 for Aβ40 and Aβ42, respectively, detects full-length Aβ1–40 and 1–42 with a sensitivity in the sub single digit fmol/ml (equivalent to single digit pg/ml) range with no cross-reactivity to APP. A combination of C-termini antibodies and an antibody against the middle region of Aβ detects mouse Aβ in non-transgenic mouse brains.
Accumulation of amyloid beta (Abeta) is a pathological hallmark of Alzheimer's disease, and lowering Abeta is a promising therapeutic approach. Intact anti‐Abeta antibodies reduce brain Abeta through ...two pathways: enhanced microglial phagocytosis and Abeta transfer from the brain to the periphery (Abeta sequestration). While activation of microglia, which is essential for microglial phagocytosis, is necessarily accompanied by undesired neuroinflammatory events, the capacity for sequestration does not seem to be linked to such effects. We and other groups have found that simple Abeta binding agents are sufficient to reduce brain Abeta through the sequestration pathway. In this study, we aimed to eliminate potentially deleterious immune activation from antibodies without affecting the ability to induce sequestration. The glycan portion of immunoglobulin is critically involved in interactions with immune effectors including the Fc receptor and complement c1q; deglycosylation eliminates these interactions, while antigen (Abeta)‐binding affinity is maintained. In this study, we investigated whether deglycosylated anti‐Abeta antibodies reduce microglial phagocytosis and neuroinflammation without altering the capacity to induce Abeta sequestration. Deglycosylated antibodies maintained Abeta binding affinity. Deglycosylated antibodies did not enhance Abeta phagocytosis or cytokine release in primary cultured microglia, whereas intact antibodies did so significantly. Intravenous injection of deglycosylated antibodies elevated plasma Abeta levels and induced Abeta sequestration to a similar or greater degree compared with intact antibodies in an Alzheimer's transgenic mouse model without or with Abeta plaque pathology. We conclude that deglycosylated antibodies effectively induced Abeta sequestration without provoking neuroinflammation; thus, these deglycosylated antibodies may be optimal for sequestration therapy for Alzheimer's disease.
Phospholipase C- eta 2 is a recently identified phospholipase C (PLC) implicated in the regulation of neuronal differentiation/maturation. PLC eta 2 activity is triggered by intracellular calcium ...mobilization and likely serves to amplify Ca2+ signals by stimulating further Ca2+ release from Ins(1,4,5)P3-sensitive stores. The role of PLC eta 2 in neuritogenesis was assessed during retinoic acid (RA)-induced Neuro2A cell differentiation. PLC eta 2 expression increased two-fold during a 4-day differentiation period. Stable expression of PLC eta 2-targetted shRNA led to a decrease in the number of differentiated cells and total length of neurites following RA-treatment. Furthermore, RA response element activation was perturbed by PLC eta 2 knockdown. Using a bacterial two-hybrid screen, we identified LIM domain kinase 1 (LIMK1) as a putative interaction partner of PLC eta 2. Immunostaining of PLC eta 2 revealed significant co-localization with LIMK1 in the nucleus and growing neurites in Neuro2A cells. RA-induced phosphorylation of LIMK1 and cAMP-responsive element-binding protein was reduced in PLC eta 2 knock-down cells. The phosphoinositide-binding properties of the PLC eta 2 PH domain, assessed using a FRET-based assay, revealed this domain to possess a high affinity toward PtdIns(3,4,5)P3. Immunostaining of PLC eta 2 together with PtdIns(3,4,5)P3 in the Neuro2A cells revealed a high degree of co-localization, indicating that PtdIns(3,4,5)P3 levels in cellular compartments are likely to be important for the spatial control of PLC eta 2 signaling. PLC eta 2 is a Ca2+-responsive phospholipase C implicated in the regulation of neuronal differentiation/maturation. Targeted knockdown of PLC eta 2 expression in retinoic acid-stimulated Neuro2A cells impacted upon neurite growth, RARE activation and LIMK1/CREB signaling. The PLC eta 2 PH domain was also found to possess a high affinity toward PtdIns(3,4,5)P3, suggesting this phospholipid to be important for the spatial control of PLC eta 2 signaling.
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