Abhik Sen

Apolipoprotein E3 (ApoE3) but Not ApoE4 Protects against Synaptic Loss through Increased Expression of Protein Kinase Cϵ

Background: ApoE4 is a genetic risk factor for sporadic AD. PKC is involved in synaptogenesis and shows abnormalities in aging and AD. Results: ApoE3 (not apoE4), acting through LRP1, protects synapses against ASPD by inducing PKCϵ. Conclusion: ApoE3 stimulates synaptogenesis and protection against ASPD by increasing PKCϵ synthesis. Significance: ApoE3 may reduce the risk for AD by stimulating PKCϵ synthesis. Synaptic loss is the earliest pathological change in Alzheimer disease (AD) and is the pathological change most directly correlated with the degree of dementia. ApoE4 is the major genetic risk factor for the age-dependent form of AD, which accounts for 95% of cases. Here we show that in synaptic networks formed from primary hippocampal neurons in culture, apoE3, but not apoE4, prevents the loss of synaptic networks produced by amyloid β oligomers (amylospheroids). Specific activators of PKCϵ, such as 8-(2-(2-pentyl-cyclopropylmethyl)-cyclopropyl)-octanoic acid methyl ester and bryostatin 1, protected against synaptic loss by amylospheroids, whereas PKCϵ inhibitors blocked this synaptic protection and also blocked the protection by apoE3. Blocking LRP1, an apoE receptor on the neuronal membrane, also blocked the protection by apoE. ApoE3, but not apoE4, induced the synthesis of PKCϵ mRNA and expression of the PKCϵ protein. Amyloid β specifically blocked the expression of PKCϵ but had no effect on other isoforms. These results suggest that protection against synaptic loss by apoE is mediated by a novel intracellular PKCϵ pathway. This apoE pathway may account for much of the protective effect of apoE and reduced risk for the age-dependent form of AD. This finding supports the potential efficacy of newly developed therapeutics for AD.

ApoE4 and Aβ Oligomers Reduce BDNF Expression via HDAC Nuclear Translocation

Apolipoprotein E4 (ApoE4) is a major genetic risk factor for several neurodegenerative disorders, including Alzheimers disease (AD). Epigenetic dysregulation, including aberrations in histone acetylation, is also associated with AD. We show here for the first time that ApoE4 increases nuclear translocation of histone deacetylases (HDACs) in human neurons, thereby reducing BDNF expression, whereas ApoE3 increases histone 3 acetylation and upregulates BDNF expression. Amyloid-β (Aβ) oligomers, which have been implicated in AD, caused effects similar to ApoE4. Blocking low-density lipoprotein receptor-related protein 1 (LRP-1) receptor with receptor-associated protein (RAP) or LRP-1 siRNA abolished the ApoE effects. ApoE3 also induced expression of protein kinase C ε (PKCε) and PKCε retained HDACs in the cytosol. PKCε activation and ApoE3 supplementation prevented ApoE4-mediated BDNF downregulation. PKCε activation also reversed Aβ oligomer- and ApoE4-induced nuclear import of HDACs, preventing the loss in BDNF. ApoE4 induced HDAC6–BDNF promoter IV binding, which reduced BDNF exon IV expression. Nuclear HDAC4 and HDAC6 were more abundant in the hippocampus of ApoE4 transgenic mice than in ApoE3 transgenic mice or wild-type controls. Nuclear translocation of HDA6 was also elevated in the hippocampus of AD patients compared with age-matched controls. These results provide new insight into the cause of synaptic loss that is the most important pathologic correlate of cognitive deficits in AD.

PKC activation during training restores mushroom spine synapses and memory in the aged rat.

Protein kinase C (PKC) ε and α activation has been implicated in synaptogenesis. We used aged rats to test whether the PKCε/α activator bryostatin and PKCε-specific activator DCP-LA combined with spatial memory training could restore mushroom dendritic spinogenesis and synaptogenesis. Compared with young rats, aged, learning-impaired rats had lower memory retention; lower densities of mushroom spines and synapses in the apical dendrites of CA1 pyramidal neurons; fewer PKCε-containing presynaptic axonal boutons; and lower activation and expression of two PKCε/α substrates, the mRNA-stabilizing protein HuD and brain-derived neurotrophic factor (BDNF). PKC activator treatment combined with spatial memory training restored mushroom spines and mushroom spine synapses; rescued PKCε/α expression and PKC/HuD/BDNF signaling; and normalized memory to the levels seen in young rats. These effects were produced by treatment with either bryostatin or the PKCε-specific activator, DCP-LA. Bryostatin also reversed alterations in GABAergic inhibitory postsynaptic currents (IPSPs) in aged, learning-impaired rats. Thus, our results support the therapeutic potential of PKC activators when added to cognitive rehabilitation for inducing mushroom spine synaptogenesis and reversing memory decline associated with aging.

Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95.

Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro. Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95S295) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin.

Bryostatin Effects on Cognitive Function and PKCɛ in Alzheimer’s Disease Phase IIa and Expanded Access Trials

Bryostatin 1, a potent activator of protein kinase C epsilon (PKCɛ), has been shown to reverse synaptic loss and facilitate synaptic maturation in animal models of Alzheimer’s disease (AD), Fragile X, stroke, and other neurological disorders. In a single-dose (25 μg/m2) randomized double-blind Phase IIa clinical trial, bryostatin levels reached a maximum at 1-2 h after the start of infusion. In close parallel with peak blood levels of bryostatin, an increase of PBMC PKCɛ was measured (p = 0.0185) within 1 h from the onset of infusion. Of 9 patients with a clinical diagnosis of AD, of which 6 received drug and 3 received vehicle within a double-blind protocol, bryostatin increased the Mini-Mental State Examination (MMSE) score by +1.83±0.70 unit at 3 h versus –1.00±1.53 unit for placebo. Bryostatin was well tolerated in these AD patients and no drug-related adverse events were reported. The 25 μg/m2 administered dose was based on prior clinical experience with three Expanded Access advanced AD patients treated with bryostatin, in which return of major functions such as swallowing, vocalization, and word recognition were noted. In one Expanded Access patient trial, elevated PKCɛ levels closely tracked cognitive benefits in the first 24 weeks as measured by MMSE and ADCS-ADL psychometrics. Pre-clinical mouse studies showed effective activation of PKCɛ and increased levels of BDNF and PSD-95. Together, these Phase IIa, Expanded Access, and pre-clinical results provide initial encouragement for bryostatin 1 as a potential treatment for AD.

Adduct formation in liquid chromatography-triple quadrupole mass spectrometric measurement of bryostatin 1

Bryostatin 1, a potential anti-Alzheimer drug, is effective at subnanomolar concentrations. Measurement is complicated by the formation of low m/z degradation products and the formation of adducts with various cations, which make accurate quantitation difficult. Adduct formation caused the sample matrix or mobile phase to partition bryostatin 1 into products of different mass. Degradation of the 927 [M+Na](+) ion to a 869m/z product was strongly influenced by ionization conditions. We validated a bryostatin 1 assay in biological tissues using capillary column HPLC with nanospray ionization (NSI) in a triple-quadrupole mass spectrometer in selected reaction monitoring (SRM) mode. Adduct formation was controlled by adding 1mM acetic acid and 0.1mM sodium acetate to the HPLC buffer, maximizing the formation of the [M+Na](+) ion. Efficient removal of contaminating cholesterol from the sample during solvent extraction was also critical. The increased sensitivity provided by NSI and capillary-bore columns and the elimination of signal partitioning due to adduct formation and degradation in the ionization source enabled a detection limit of 1×10(-18)mol of bryostatin 1 and a LLOQ of 3×10(-18)mol from 1μl of sample. Bryostatin 1 at low pmol/l concentrations enabled measurement in brain and other tissues without the use of radioactive labels. Despite bryostatin 1s high molecular weight, considerable brain access was observed, with peak brain concentrations exceeding 8% of the peak blood plasma concentrations. Bryostatin 1 readily crosses the blood-brain barrier, reaching peak concentrations of 0.2nM, and specifically activates and translocates brain PKCɛ.

Hippocampal microvasculature changes in association with oxidative stress in Alzheimer's disease

ABSTRACT Vascular endothelial dysfunction is a primary phenotype of aging, and microvascular (MV) lesion is mainly associated with Alzheimers disease (AD). Here we have studied the correlation of MV wall thickness and CA1 pyramidal neuronal pathology in autopsy‐confirmed AD brains. Both hyaline (h‐MV) and increased cell number (c‐MV) associated MV wall thickening was found in age‐matched control (AC) hippocampus without significant change in A&bgr; level (Braak stages 0‐III). AC neurons neighboring the h‐MV showed lower levels of oxidative DNA/RNA damage and A&bgr; precursor protein (APP), while the neurons around c‐MV showed higher oxidative DNA/RNA damage with increased APP expression. Neurons in AC hippocampus without MV wall thickening (thin wall) showed increased DNA/RNA damage and APP levels compared to AC cases with h‐MV and c‐MV walls. In the AD hippocampus neurons neighboring h‐MV walls showed increased levels of A&bgr; and decreased number of dendritic spines (at Braak stages IV‐VI). C‐MV neighboring neurons in the AD cases showed higher levels of DNA/RNA damage with increased APP at stages II ‐ III, followed by lower levels of oxidative DNA/RNA damage, decreased APP and increased A&bgr; levels with loss of dendritic spines at stages IV‐VI. Prolonged treatment of primary human fetal hippocampal neurons with tert‐butyl hydroperoxide (TBHP) induced oxidative DNA damage with a sustained increase in APP. A&bgr; increased rapidly and then decreased overtime. Short‐term TBHP treated neurons showed lower levels of superoxide (O2• −) without significant DNA damage. Short‐term TBHP treatment induced a gradual decrease in APP but an increase in A&bgr; levels over time. In conclusion this study indicates that AD hippocampus at Braak stages II‐III are characterized by strong oxidative DNA/RNA damage with increased APP in neurons associated with c‐MV, while stages IV‐VI are characterized by a slow increase in A&bgr; in neurons neighboring both h‐MV and c‐MV. HIGHLIGHTSHyaline (h‐) & increased cell number (c‐) microvascular (MV) wall thickening.Non‐AD thin wall MV neurons have higher oxidative damage & APP than h‐ or c‐MV neurons.Non‐AD h‐MV neurons have lower oxidative stress marker & APP than c‐MV neurons.Strong oxidative damage & increased APP in AD neuron with c‐MV (Braak stages II‐III).Mild oxidative stress & increased A&bgr; in AD neurons with h‐MV or c‐MV (stages IV‐VI).