Ottavio V. Vitolo

Endoplasmic Reticulum Stress and the Unfolded Protein Response in Cellular Models of Parkinson's Disease

6-Hydroxydopamine, 1-methyl-4-phenyl-pyridinium (MPP+), and rotenone cause the death of dopaminergic neurons in vitro and in vivo and are widely used to model Parkinsons disease. To identify regulated genes in such models, we performed serial analysis of gene expression on neuronal PC12 cells exposed to 6-hydroxydopamine. This revealed a striking increase in transcripts associated with the unfolded protein response. Immunoblotting confirmed phosphorylation of the key endoplasmic reticulum stress kinases IRE1α and PERK (PKR-like ER kinase) and induction of their downstream targets. There was a similar response to MPP+ and rotenone, but not to other apoptotic initiators. As evidence that endoplasmic reticulum stress contributes to neuronal death, sympathetic neurons from PERK null mice in which the capacity to respond to endoplasmic reticulum stress is compromised were more sensitive to 6-hydroxydopamine. Our findings, coupled with evidence from familial forms of Parkinsons disease, raise the possibility of widespread involvement of endoplasmic reticulum stress and the unfolded protein response in the pathophysiology of this disease.

Amyloid β-peptide inhibition of the PKA/CREB pathway and long-term potentiation: Reversibility by drugs that enhance cAMP signaling

Changes in hippocampal function seem critical for cognitive impairment in Alzheimers disease (AD). Although there is eventual loss of synapses in both AD and animal models of AD, deficits in spatial memory and inhibition of long-term potentiation (LTP) precede morphological alterations in the models, suggesting earlier biochemical changes in the disease. In the studies reported here we demonstrate that amyloid β-peptide (Aβ) treatment of cultured hippocampal neurons leads to the inactivation of protein kinase A (PKA) and persistence of its regulatory subunit PKAIIα. Consistent with this, CREB phosphorylation in response to glutamate is decreased, and the decrease is reversed by rolipram, a phosphodiesterase inhibitor that raises cAMP and leads to the dissociation of the PKA catalytic and regulatory subunits. It is likely that a similar mechanism underlies Αβ inhibition of LTP, because rolipram and forskolin, agents that enhance the cAMP-signaling pathway, can reverse this inhibition. This reversal is blocked by H89, an inhibitor of PKA. These observations suggest that Αβ acts directly on the pathways involved in the formation of late LTP and agents that enhance the cAMP/PKA/CREB-signaling pathway have potential for the treatment of AD.

Functional Alterations in Memory Networks in Early Alzheimer’s Disease

The hallmark clinical symptom of early Alzheimer’s disease (AD) is episodic memory impairment. Recent functional imaging studies suggest that memory function is subserved by a set of distributed networks, which include both the medial temporal lobe (MTL) system and the set of cortical regions collectively referred to as the default network. Specific regions of the default network, in particular, the posteromedial cortices, including the precuneus and posterior cingulate, are selectively vulnerable to early amyloid deposition in AD. These regions are also thought to play a key role in both memory encoding and retrieval, and are strongly functionally connected to the MTL. Multiple functional magnetic resonance imaging (fMRI) studies during memory tasks have revealed alterations in these networks in patients with clinical AD. Similar functional abnormalities have been detected in subjects at-risk for AD, including those with genetic risk and older individuals with mild cognitive impairment. Recently, we and other groups have found evidence of functional alterations in these memory networks even among cognitively intact older individuals with occult amyloid pathology, detected by PET amyloid imaging. Taken together, these findings suggest that the pathophysiological process of AD exerts specific deleterious effects on these distributed memory circuits, even prior to clinical manifestations of significant memory impairment. Interestingly, some of the functional alterations seen in prodromal AD subjects have taken the form of increases in activity relative to baseline, rather than a loss of activity. It remains unclear whether these increases in fMRI activity may be compensatory to maintain memory performance in the setting of early AD pathology or instead, represent evidence of excitotoxicity and impending neuronal failure. Recent studies have also revealed disruption of the intrinsic connectivity of these networks observable even during the resting state in early AD and asymptomatic individuals with high amyloid burden. Research is ongoing to determine if these early network alterations will serve as sensitive predictors of clinical decline, and eventually, as markers of pharmacological response to potential disease-modifying treatments for AD.

Persistent improvement in synaptic and cognitive functions in an Alzheimer mouse model after rolipram treatment

Evidence suggests that Alzheimer disease (AD) begins as a disorder of synaptic function, caused in part by increased levels of amyloid beta-peptide 1-42 (Abeta42). Both synaptic and cognitive deficits are reproduced in mice double transgenic for amyloid precursor protein (AA substitution K670N,M671L) and presenilin-1 (AA substitution M146V). Here we demonstrate that brief treatment with the phosphodiesterase 4 inhibitor rolipram ameliorates deficits in both long-term potentiation (LTP) and contextual learning in the double-transgenic mice. Most importantly, this beneficial effect can be extended beyond the duration of the administration. One course of long-term systemic treatment with rolipram improves LTP and basal synaptic transmission as well as working, reference, and associative memory deficits for at least 2 months after the end of the treatment. This protective effect is possibly due to stabilization of synaptic circuitry via alterations in gene expression by activation of the cAMP-dependent protein kinase (PKA)/cAMP regulatory element-binding protein (CREB) signaling pathway that make the synapses more resistant to the insult inflicted by Abeta. Thus, agents that enhance the cAMP/PKA/CREB pathway have potential for the treatment of AD and other diseases associated with elevated Abeta42 levels.

Ubiquitin Hydrolase Uch-L1 Rescues β-Amyloid-Induced Decreases in Synaptic Function and Contextual Memory

The neuronal ubiquitin/proteasomal pathway has been implicated in the pathogenesis of Alzheimers disease (AD). We now show that a component of the pathway, ubiquitin C-terminal hydrolase L1 (Uch-L1), is required for normal synaptic and cognitive function. Transduction of Uch-L1 protein fused to the transduction domain of HIV-transactivator protein (TAT) restores normal enzymatic activity and synaptic function both in hippocampal slices treated with oligomeric Abeta and in the APP/PS1 mouse model of AD. Moreover, intraperitoneal injections with the fusion protein improve the retention of contextual learning in APP/PS1 mice over time. The beneficial effect of the Uch-L1 fusion protein is associated with restoration of normal levels of the PKA-regulatory subunit IIalpha, PKA activity, and CREB phosphorylation.

Dendrite and dendritic spine alterations in Alzheimer models.

Synaptic damage and loss are factors that affect the degree of dementia experienced in Alzheimer disease (AD) patients. Multicolor DiOlistic labeling of the hippocampus has been undertaken which allows the full dendritic arbor of targeted neurons to be imaged. Using this labeling technique the neuronal morphology of two transgenic mouse lines (J20 and APP/PS1) expressing mutant forms of the Amyloid Precursor Protein (APP), at various ages, have been visualized and compared to Wild Type (WT) littermate controls. Swollen bulbous dystrophic neurites with loss of spines were apparent in the transgenic animals. Upon quantification, statistically significant reductions in the number of spines and total dendrite area was observed in both transgenic mouse lines at 11 months of age. Similar morphological abnormalities were seen in human AD hippocampal tissue both qualitatively and quantitatively. Immunohistochemistry and DiOlistic labeling was combined so that Aβ plaques were imaged in relation to the dendritic trees. No preferential localization of these abnormal dystrophic neurites was seen in regions with plaques. DiI labeled reative astrocytes were often apparent in close proximity to Aβ plaques.

Amyloid-β Peptide Inhibits Activation of the Nitric Oxide/cGMP/cAMP-Responsive Element-Binding Protein Pathway during Hippocampal Synaptic Plasticity

Amyloid-β (Aβ), a peptide thought to play a crucial role in Alzheimers disease (AD), has many targets that, in turn, activate different second-messenger cascades. Interestingly, Aβ has been found to markedly impair hippocampal long-term potentiation (LTP). To identify a new pathway that might be responsible for such impairment, we analyzed the role of the nitric oxide (NO)/soluble guanylyl cyclase (sGC)/cGMP/cGMP-dependent protein kinase (cGK)/cAMP-responsive element-binding protein (CREB) cascade because of its involvement in LTP. The use of the NO donor 2-(N,N-dethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO), the sGC stimulator 3-(4-amino-5-cyclopropylpyrimidine-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine, or the cGMP-analogs 8-bromo-cGMP and 8-(4-chlorophenylthio)-cGMP reversed the Aβ-induced impairment of CA1-LTP through cGK activation. Furthermore, these compounds reestablished the enhancement of CREB phosphorylation occurring during LTP in slices exposed to Aβ. We also found that Aβ blocks the increase in cGMP immunoreactivity occurring immediately after LTP and that DEA/NO counteracts the effect of Aβ. These results strongly suggest that, when modulating hippocampal synaptic plasticity, Aβ downregulates the NO/cGMP/cGK/CREB pathway; thus, enhancement of the NO/cGMP signaling may provide a novel approach to the treatment of AD and other neurodegenerative diseases with elevated production of Aβ.

Amyloid β peptide adversely affects spine number and motility in hippocampal neurons

Elevated levels of amyloid-beta peptide (Abeta) are found in Downs syndrome patients and alter synaptic function during the early stages of Alzheimers disease. Dendritic spines, sites of most excitatory synaptic contacts, are considered to be an important locus for encoding synaptic plasticity. We used time-lapse two-photon imaging of hippocampal pyramidal neurons in organotypic slices to study the effects of Abeta on the development of dendritic spines. We report that exposure of hippocampal neurons to sub-lethal levels of Abeta decreased spine density, increased spine length and subdued spine motility. The effect of Abeta on spine density was reversible. Moreover, Abetas effect on dendritic spine density was blocked by rolipram, a phosphodiesterase type IV inhibitor, suggesting the involvement of a cAMP dependent pathway. These findings raise the possibility that Abeta-induced spine alterations could underlie the cognitive defects in Alzheimers disease and Down syndrome.

Adenosine and ADP prevent apoptosis in cultured rat cerebellar granule cells

Cerebellar granule cells (CGCs) explanted in vitro undergo death via apoptosis when the concentration of potassium is shifted from 25 mM to 5 mM. We report that adenosine and ADP, which act as neurotransmitters and neuromodulators in the brain, exert in cultured cerebellar granule cells a specific and marked antiapoptotic action with half-maximal effect in the 10-100 microM range. The action of adenosine is partly inhibited by the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and is mimicked by the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA), while ADP effect, that is completely blocked by the P2x, P2y receptors noncompetitive antagonist suramine, is restored in the presence of the selective P2x purinoceptors agonist beta, gamma-methylene-L-ATP. These findings demonstrate that adenosine and ADP markedly inhibit the program of cell death in cerebellar granule cells and suggest that such an action is mediated via interaction with, respectively, A1 and P2x receptors.

Protection against β-amyloid induced abnormal synaptic function and cell death by Ginkgolide J.

A new Ginkgo biloba extract P8A (TTL), 70% enriched with terpene trilactones, prevents A beta(1-42) induced inhibition of long-term potentiation in the CA1 region of mouse hippocampal slices. This neuroprotective effect is attributed in large part to ginkgolide J that completely replicates the effect of the extract. Ginkgolide J is also capable of inhibiting cell death of rodent hippocampal neurons caused by A beta(1-42). This beneficial and multi-faceted mode of action of the ginkgolide makes it a new and promising lead in designing therapies against Alzheimers disease.