Martin A. Bruno

Translational control of hippocampal synaptic plasticity and memory by the eIF2α kinase GCN2

Studies on various forms of synaptic plasticity have shown a link between messenger RNA translation, learning and memory. Like memory, synaptic plasticity includes an early phase that depends on modification of pre-existing proteins, and a late phase that requires transcription and synthesis of new proteins. Activation of postsynaptic targets seems to trigger the transcription of plasticity-related genes. The new mRNAs are either translated in the soma or transported to synapses before translation. GCN2, a key protein kinase, regulates the initiation of translation. Here we report a unique feature of hippocampal slices from GCN2-/- mice: in CA1, a single 100-Hz train induces a strong and sustained long-term potentiation (late LTP or L-LTP), which is dependent on transcription and translation. In contrast, stimulation that elicits L-LTP in wild-type slices, such as four 100-Hz trains or forskolin, fails to evoke L-LTP in GCN2-/- slices. This aberrant synaptic plasticity is mirrored in the behaviour of GCN2-/- mice in the Morris water maze: after weak training, their spatial memory is enhanced, but it is impaired after more intense training. Activated GCN2 stimulates mRNA translation of ATF4, an antagonist of cyclic-AMP-response-element-binding protein (CREB). Thus, in the hippocampus of GCN2-/- mice, the expression of ATF4 is reduced and CREB activity is increased. Our study provides genetic, physiological, behavioural and molecular evidence that GCN2 regulates synaptic plasticity, as well as learning and memory, through modulation of the ATF4/CREB pathway.

Amyloid β-Induced Nerve Growth Factor Dysmetabolism in Alzheimer Disease

We previously reported that the precursor form of nerve growth factor (pro-NGF) and not mature NGF is liberated in the CNS in an activity-dependent manner, and that its maturation and degradation occur in the extracellular space by the coordinated action of proteases.Here, we present evidence of diminished conversion of pro-NGF to its mature form and of greater NGF degradation in Alzheimer disease (AD) brain samples compared with controls. These alterations of the NGF metabolic pathway likely resulted in the increased pro-NGF levels. The pro-NGF was largely in a peroxynitrited form in the AD samples. Intrahippocampal injection of amyloid-&bgr; oligomers provoked similar upregulation of pro-NGF in naive rats that wasaccompanied by evidence of microglial activation (CD40), increased levels of inducible nitric oxide synthase, and increased activity of the NGF-degrading enzyme matrix metalloproteinase 9. The elevated inducible nitric oxide synthase provoked the generation of biologically inactive, peroxynitrite-modified pro-NGF in amyloid-&bgr; oligomer-injected rats. These parameters were corrected by minocycline treatment. Minocycline also diminished altered matrix metalloproteinase 9, inducible nitric oxide synthase, and microglial activation (CD40); improved cognitive behavior; and normalized pro-NGF levels in a transgenic mouse AD model. The effects of amyloid-&bgr; amyloid CNS burden on NGF metabolism may explain the paradoxical upregulation of pro-NGF in AD accompanied by atrophy of forebrain cholinergic neurons.

Increased matrix metalloproteinase 9 activity in mild cognitive impairment.

Nerve growth factor (NGF)-dependent cholinergic basal forebrain neurons degenerate during the progression of Alzheimer disease (AD). Elevated proNGF and reduced levels of the TrkA high-affinity NGF receptor occur in prodromal and advanced stages of AD. We recently described a protease cascade responsible for the conversion of proNGF to mature NGF (mNGF) in which matrix metalloproteinase 9 (MMP-9) degrades mNGF in the extracellular space. To determine whether this proteolytic cascade is altered during the progression of AD, we examined human frontal and parietal cortex tissues from aged subjects with a clinical diagnosis of AD, mild cognitive impairment, or no cognitive impairment. The analysis demonstrated greater MMP-9 activity in both AD and mild cognitive impairment compared with no cognitive impairment brain samples (p < 0.01), which supports the notion that a metabolic failure in the NGF-maturation/degradation pathway may be associated with an exacerbated degradation of mNGF in the cerebral cortex in early AD. Moreover, there were inverse correlations between Global Cognitive Score and Mini-Mental State Examination score and MMP-9 activity. These findings suggest that a reduction in mNGF as a consequence of MMP-9-mediated degradation may in part underlie the pathogenesis of cognitive deficits in mild cognitive impairment and AD.

Cholinergic Involvement in Alzheimer’s Disease. A Link with NGF Maturation and Degradation

Basal forebrain cholinergic neurons are highly dependent on nerve growth factor (NGF) supply for the maintenance of their cholinergic phenotype as well as their cholinergic synaptic integrity. The precursor form of NGF, proNGF, abounds in the CNS and is highly elevated in Alzheimer’s disease. In order to obtain a deeper understanding of the NGF biology in the CNS, we have performed a series of ex vivo and in vivo investigations to elucidate the mechanisms of release, maturation and degradation of this neurotrophin. In this short review, we describe this NGF metabolic pathway, its significance for the maintenance of basal cholinergic neurons, and its dysregulation in Alzheimer’s disease. We are proposing that the conversion of proNGF to mature NGF occurs in the extracellular space by the coordinated action of zymogens, convertases, and endogenous regulators, which are released in the extracellular space in an activity-dependent fashion. We further discuss our findings of a diminished conversion of the NGF precursor molecule to its mature form in Alzheimer’s disease as well as an augmented degradation of mature NGF. These combined effects on NGF metabolism would explain the well-known cholinergic atrophy found in Alzheimer’s disease and would offer new therapeutic opportunities aimed at correcting the NGF dysmetabolism along with Aβ-induced inflammatory responses.

Long-Lasting Rescue of Age-Associated Deficits in Cognition and the CNS Cholinergic Phenotype by a Partial Agonist Peptidomimetic Ligand of TrkA

Previously, we developed a proteolytically stable small molecule peptidomimetic termed D3 as a selective ligand of the extracellular domain of the TrkA receptor for the NGF. Ex vivo D3 was defined as a selective, partial TrkA agonist. Here, the in vivo efficacy of D3 as a potential therapeutic for cholinergic neurons was tested in cognitively impaired aged rats, and we compared the consequence of partial TrkA activation (D3) versus full TrkA/p75 activation (NGF). We show that in vivo D3 binds to TrkA receptors and affords a significant and long-lived phenotypic rescue of the cholinergic phenotype both in the cortex and in the nucleus basalis. The cholinergic rescue was selective and correlates with a significant improvement of memory/learning in cognitively impaired aged rats. The effects of the synthetic ligand D3 and the natural ligand NGF were comparable. Small, proteolytically stable ligands with selective agonistic activity at a growth factor receptor may have therapeutic potential for neurodegenerative disorders.

Engagement of the PFC in consolidation and recall of recent spatial memory

The standard model of system consolidation proposes that memories are initially hippocampus dependent and become hippocampus independent over time. Previous studies have demonstrated the involvement of the medial prefrontal cortex (mPFC) in the retrieval of remote memories. The transformations required to make a memory undergo systems consolidation are thought to require synaptic plasticity. In this study, we investigated the participation of the mitogen-activated protein kinase (MAPK)/ERK pathway in acquisition, memory consolidation, and recent memory recall of the Morris water maze (MWM) task using a 1-d training protocol. To this end, bilateral injections of the MEK inhibitor U0126 into the rat mPFC were performed. The injection of the MEK inhibitor in the mPFC did not affect the acquisition of the MWM. However, MEK inhibitor resulted in impairments on recent memory retrieval either when applied at the end of the learning phase (memory consolidation) or prior to the retention test. The results strongly support the concept that recently acquired and consolidated spatial memories require the mPFC, and that local activation of the MAPK/ERK pathway in the mPFC is necessary for the consolidation and recall of recent memories.

Intracellular Aβ-oligomers and early inflammation in a model of Alzheimer's disease

Lifelong use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to diminish the incidence of Alzheimers disease (AD), suggesting a key role of inflammation in early stages of the pathology. While amyloid plaque-associated inflammation has been extensively studied in human and animal models, little is known about the inflammatory process prior to plaque deposition, i.e., in preclinical stages of AD. In this study we investigated microglial and neuronal inflammatory markers in preplaque transgenic McGill-Thy1-APP mice. We found evidence that prior to plaque deposition classical markers of microglial activation such as major histocompatibility complex class II (MHC-II), inducible nitric oxide synthase (i-NOS), and CD40 are already upregulated in the hippocampus of transgenic mice. Microglial cells from transgenic mice in the preplaque stage displayed intermediately activated morphology and appeared to be recruited toward intracellular amyloid-β peptide (Aβ)-oligomer burdened neurons. The inducible, neuron-specific cyclooxygenase 2 (COX-2) enzyme was found to be upregulated and specifically expressed by neurons in close relationship with Aβ-bearing cells, at this early stage of the AD-like pathology. Our study suggests that neuroinflammation might be one of the earliest pathological responses to intracellular accumulation of Aβ-oligomers.

The Failure in NGF Maturation and its Increased Degradation as the Probable Cause for the Vulnerability of Cholinergic Neurons in Alzheimer’s Disease

This short review discusses the arguments to consider the dismetabolism of the pathway responsible for both the maturation and degradation of NGF as the culprit of vulnerability of the forebrain cholinergic system to the Alzheimer’s disease neuropathology. This summary includes information regarding a novel metabolic cascade converting Pro-NGF to mature NGF in the extracellular space and its ultimate degradation by a metalloprotease. It also describes how this pathway is altered in Alzheimer’s disease with the consequential CNS accumulation of proNGF and impairment in the formation of NGF along with increased degradation of this key trophic factor. This metabolic scenario in Alzheimer’s disease should result in the failure of NGF trophic support to forebrain cholinergic neurons and thus explaining the vulnerability of these neurons in this neurodegenerative condition.

Impact of the NGF Maturation and Degradation Pathway on the Cortical Cholinergic System Phenotype

Cortical cholinergic atrophy plays a significant role in the cognitive loss seen with aging and in Alzheimers disease (AD), but the mechanisms leading to it remain unresolved. Nerve growth factor (NGF) is the neurotrophin responsible for the phenotypic maintenance of basal forebrain cholinergic neurons in the mature and fully differentiated CNS. In consequence, its implication in cholinergic atrophy has been suspected; however, no mechanistic explanation has been provided. We have previously shown that the precursor of NGF (proNGF) is cleaved extracellularly by plasmin to form mature NGF (mNGF) and that mNGF is degraded by matrix metalloproteinase 9. Using cognitive-behavioral tests, Western blotting, and confocal and electron microscopy, this study demonstrates that a pharmacologically induced chronic failure in extracellular NGF maturation leads to a reduction in mNGF levels, proNGF accumulation, cholinergic degeneration, and cognitive impairment in rats. It also shows that inhibiting NGF degradation increases endogenous levels of the mature neurotrophin and increases the density of cortical cholinergic boutons. Together, the data point to a mechanism explaining cholinergic loss in neurodegenerative conditions such as AD and provide a potential therapeutic target for the protection or restoration of this CNS transmitter system in aging and AD.

Early-Stage Inflammation and Experimental Therapy in Transgenic Models of the Alzheimer-Like Amyloid Pathology

Background: Intracellular accumulation of β-amyloid (Aβ) is one of the early features in the neuropathology of Alzheimer’s disease (AD) and Down’s syndrome. This can be reproduced in cell and transgenic animal models of the AD-like amyloid pathology. In a transgenic rat model, our lab has previously shown that the intracellular accumulation of Aβ is sufficient to provoke cognitive impairments and biochemical alterations in the cerebral cortex and hippocampus in the absence of amyloid plaques. Objective: To investigate an early, pre-plaque inflammatory process in AD-like transgenic models and establish whether the neurotoxic effects of Aβ oligomers and proinflammatory responses can be arrested with minocycline. Methods: For these studies, we used naïve mice and transgenic animal models of the AD-like amyloid pathology and applied neurochemical, immunohistochemical and behavioral experimental approaches. Results: In the early stages of the AD-like amyloid pathology, intracellular Aβ oligomers accumulate within neurons of the cerebral cortex and hippocampus. Coincidental with this, behavioral impairments occur prior to the appearance of amyloid plaques, together with an upregulation of MHC-II, i-NOS and COX-2, well-known proinflammatory markers. Treatment with minocycline corrected behavioral impairments, lowered inflammatory markers and levels of Aβ trimers. Conclusion: A pharmacological approach targeting the early neuroinflammatory effects of Aβ might be a promising strategy to prevent or delay the onset of AD.