Jarin Hongpaisan

PKC ε Activation Prevents Synaptic Loss, Aβ Elevation, and Cognitive Deficits in Alzheimer's Disease Transgenic Mice

Among the pathologic hallmarks of Alzheimers disease (AD) neurodegeneration, only synaptic loss in the brains of AD patients closely correlates with the degree of dementia in vivo. Here, we describe a molecular basis for this AD loss of synapses: pathological reduction of synaptogenic PKC isozymes and their downstream synaptogenic substrates, such as brain-derived neurotrophic factor. This reduction, particularly of PKC α and ε, occurs in association with elevation of soluble β amyloid protein (Aβ), but before the appearance of the amyloid plaques or neuronal loss in the Tg2576 AD transgenic mouse strain. Conversely, treatment of the Tg2576 mouse brain with the PKC activator, bryostatin-1, restores normal or supranormal levels of PKC α and ε, reduces the level of soluble Aβ, prevents and/or reverses the loss of hippocampal synapses, and prevents the memory impairment observed at 5 months postpartum. Similarly, the PKC ε-specific activator, DCP-LA, effectively prevents synaptic loss, amyloid plaques, and cognitive deficits (also prevented by bryostatin-1) in the much more rapidly progressing 5XFAD transgenic strain. These results suggest that synaptic loss and the resulting cognitive deficits depend on the balance between the lowering effects of Aβ on PKC α and ε versus the lowering effects of PKC on Aβ in AD transgenic mice.

Strong Calcium Entry Activates Mitochondrial Superoxide Generation, Upregulating Kinase Signaling in Hippocampal Neurons

Large increases in cytosolic free Ca2+ ([Ca2+]i) activate several kinases that are important for neuronal plasticity, including Ca2+/calmodulin-dependent kinase II (CaMKII), protein kinase A (PKA), and protein kinase C (PKC). Because it is also known, mainly in non-neuronal systems, that superoxide radicals (O2-) activate these (and other) kinases and because O2- generation by mitochondria is in part [Ca2+]i dependent, we examined in hippocampal neurons the relationship between Ca2+ entry, O2- production, and kinase activity. We found that, after large stimulus-induced [Ca2+]i increases, O2- selectively produced by mitochondria near plasmalemmal sites of Ca2+ entry acts as a modulator to upregulate the two kinases, namely, CaMKII and PKA, whose activities are directly or indirectly phosphorylation dependent. The common mechanism involves O2- inhibition of inactivating protein phosphatases. Conversely, because small [Ca2+]i increases do not promote mitochondrial respiration and O2- generation, weak stimuli favor enhanced phosphatase activity, which therefore leads to suppressed kinase activity. Enhanced O2- production also promoted PKC activity but by a phosphatase-independent pathway. These results suggest that Ca2+-dependent upregulation of mitochondrial O2- production may be a general mechanism for linking Ca2+ entry to enhanced kinase activity and therefore to synaptic plasticity. This mechanism also represents yet another way that mitochondria, acting as calcium sensors, can play a role in neuronal signal transduction.

Calcium-dependent mitochondrial superoxide modulates nuclear CREB phosphorylation in hippocampal neurons

We report evidence that mitochondrially produced superoxide (O(2)(-)) is involved in signaling in hippocampal neurons by examining the relationship between strong but physiological increases in cytosolic free Ca(2+), mitochondrial calcium accumulation, O(2)(-) production, and CREB phosphorylation. Strong depolarization-induced Ca(2+) entry through NMDA or L-type Ca(2+) channels evoked large Ca(2+) transients, a sustained increase in O(2)(-), and a large rise in nuclear CaM and pCREB. Under these conditions, inhibition of mitochondrial Ca(2+) uptake and consequent O(2)(-) production suppressed Ca(2+) entry-induced pCREB elevation, indicating that O(2)(-) produced by mitochondria supports CREB phosphorylation. Similarly, inhibiting mitochondrial respiration blocked O(2)(-) production and also depressed the elevation of pCREB. Blocking calcineurin reversed this depression. We conclude that strong Ca(2+) entry promotes mitochondrial calcium accumulation and the subsequent enhancement of mitochondrial O(2)(-) production, which in turn prolongs the lifetime of pCREB by suppressing calcineurin-dependent pCREB dephosphorylation.

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.

Effects of chronic bryostatin-1 on treatment-resistant depression in rats

Abstract Despite over a half‐centurys intensive research worldwide, the currently available antidepressants remain sub‐optimal. Therapeutic options for treatment‐resistant depression, for instance, are rather limited. Here, we found that rats exhibited a lasting treatment‐resistant depressive immobility in response to open space swim test at a high intensity of induction. The induced depressive behavior is associated with a dramatic impairment in spatial learning and memory. Both the depressive immobility and impairment in spatial learning and memory are sensitive to a period of chronic treatment with bryopstatin‐1, a relatively selective activator of protein kinase C&egr;. Bryostatin‐1‐like analogues therefore might have therapeutic values for the treatment of treatment‐resistant depression.

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).