Neurochemical Research

, Volume 32, Issue 6, pp 1041–1045

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


    • Departments of Pharmacology & TherapeuticsMcGill University
    • Anatomy and Cell BiologyMcGill University
    • Neurology and NeurosurgeryMcGill University
  • Martin A. Bruno
    • Departments of Pharmacology & TherapeuticsMcGill University
Original Paper

DOI: 10.1007/s11064-006-9270-0

Cite this article as:
Cuello, A.C. & Bruno, M.A. Neurochem Res (2007) 32: 1041. doi:10.1007/s11064-006-9270-0


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.


Alzheimer’s diseasePro-NGFNGFMetalloproteaseCholinergic neuronsTrophic factorNeurodegeneration


It is for these authors a great privilege to contribute to a tribute to Moussa Youdim who has been one of the most influential pharmacologists in the search for novel therapeutics to revert the degenerative process of dopaminergic neurons in Parkinson disease. For one of us (A. Claudio Cuello), this is particularly so because their paths have crossed in Oxford and McGill at different times, and because they share strong scientific interests and a long friendship; and for the co-author (Martin A. Bruno), it is a privilege because he has benefited from Moussa’s inspirational lectures at McGill. Moussa’s broad interests include the cholinergic system and Alzheimer’s disease. The cholinergic involvement in Alzheimer’s disease (AD) pathology is well established and need not be fully reviewed here. It may suffice to say that several decades ago, a significant loss of neurochemical cholinergic markers in the cerebral cortex of AD brains was reported by Davis and collaborators, and Bowen and collaborators [1, 2]. Further to this, Whitehouse et al. [3] reported the loss of cholinergic neurons presumptively in the nucleus magnocellularis of Meynert, i.e., nucleus basalis, in post-mortem samples of AD sufferers (no cholinergic markers were yet available). This prompted the formulation of the so-called “cholinergic hypothesis of AD”, with the knowledge of the central role of cholinergic neurons in memory mechanisms. The hypothesis was equivalent to the anterograde loss of dopaminergic neurons in Parkinson’s disease, assuming an anterograde loss of cholinergic neurons in AD. We instead proposed in 1984 [4] that the cholinergic involvement in AD was secondary to cortical lesion (retrograde degeneration) based on our experimental evidence for basalis nucleus cell shrinkage following stroke-type cortical lesions [5]. Since those early studies, we have learned a great deal about the relationship between the cortical amyloid burden and the cholinergic synaptic involvement thanks to the development of transgenic animal models of the AD-like amyloid pathology. Thus, we have been able to establish a pathology and time-dependent involvement of cortical cholinergic synaptic losses and generation of cholinergic dystrophic neurites in such models [6]. In brief, these investigations clearly signal the preferential vulnerability of cortical terminations of cholinergic projections of the basal forebrain in the presence of progressive Aβ-induced pathology, a finding which is consistent with the abundant literature from neurochemical studies in AD human brain material. What is important, however, from the transgenic studies is that the Aβ burden per se, without any added factor (e.g., tau pathology), is sufficient to replicate the vulnerability of cortical cholinergic projections observed in the AD brain.

Forebrain cholinergic neurons, as is well established, are highly dependent of the endogenous supply of NGF throughout life. This concept could be extended to the day to day supply of NGF in the cerebral cortex. This is illustrated in experiments in which blocking endogenous NGF in the cerebral cortex with monoclonal antibodies or TrkA antagonist results in the rapid disappearance of pre-existing cholinergic synapses [7]. Would a failure in NGF supply in AD therefore explain the remarkable vulnerability of the NGF-dependent forebrain cholinergic neurons? This issue was investigated early by many authors who found no evidence for such a deficiency. In most cases, the AD brain revealed normal or augmented NGF mRNA [8, 9] and normal or augmented NGF [1012]. More recently, the work of Fahnestock and collaborators [13] clearly indicates that in AD there is an unequivocal up-regulation of the NGF precursor molecule, proNGF. Moreover, it has been proposed that, in the adult CNS, proNGF expression is up-regulated following CNS lesions, probably contributing to cell death through p75NTR and sortilin [14, 15].

For over two decades, it has been assumed that the mature NGF form accounts for the neurotrophin’s biological activity, including cell survival, neurite outgrowth and neuronal differentiation. The realization that proNGF might play a biological role in the CNS raised questions regarding the regulatory mechanisms leading to its release, as well as the control of the proNGF to NGF ratio and ultimately the degradation of the NGF molecule. To answer these questions, we embarked on a series of in vitro and in vivo studies aimed at elucidating the preferential NGF form released from the cerebral cortex and the pathway leading to NGF maturation and degradation. These studies have revealed that proNGF is the main releasable form of the neurotrophin and that the maturation and degradation of NGF largely occurs in the extracellular space with the involvement of a complex protease cascade [16].

In this regard, we have gathered experimental evidence that the protease cascade responsible for both the maturation of NGF from proNGF to mature NGF is released from cortical neurons, along with proNGF, in an activity-dependent manner. In brief, we have proposed that the conversion of proNGF to NGF takes place in the extracellular space, as opposed to the prevalent view that the NGF maturation occurs intracellularly. This extracellular conversion of Pro-NGF into mature NGF takes place by the activation plasmin from plasminogen with the participation of tissue plasminogen activator (tPA), a mechanism regulated by neuroserpin [16]. Likewise, we have proposed that the degradation and consequent inactivation of the NGF which is not bound to receptors or internalized also takes place in the extracellular space. This process is mediated by the activation of the precursor of matrix metalloproteinase 9 (MMP-9) by plasmin and its activity regulated by the tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) [16]. As mentioned above, we have shown that this novel protease cascade, endogenous protease regulators and the NGF precursor, proNGF, are simultaneously released upon neuronal stimulation. From these observations, some fundamental issues are derived: (1) that most, if not all, of the radioimmunoassable NGF in cortical tissues from past investigations demonstrate proNGF and not mature NGF, (2) the precursor form of NGF is delivered “on demand” in a neuronal activity-dependent manner and, (3) that the reason why the biologically active mature NGF has been so elusive is because the newly generated NGF rapidly binds the TrkA receptors at high affinity while the remnant NGF is promptly degraded by the activated MMP-9. The occurrence of such a metabolic cascade for the conversion and ulterior degradation of NGF is perhaps one of the finest examples that metabolic protein/enzymatic complexes do not act at random but rather they are coordinated to be synthesized and delivered to their site of action in functional clusters [16]. This metabolic pathway and its potential significance for forebrain cholinergic are schematically represented in Fig. 1.
Fig. 1

Maturation and degradation of NGF (A) Schematic representation of the cascade responsible for the processing of the activity-dependent released of proNGF, its conversion to mature NGF (mNGF) and its degradation by the activated matrix metallo-protease 9 (MMP-9) [16].The activation of this pathway should favor cholinergic function, which is known to switch the APP (Amyloid Precursor Protein) towards a non-amyloidogenic (α-secretase) pathway via the activation of muscarinic receptors M1 and M3. (B) Dysregulation of the cascade responsible for the processing and degradation of NGF observed in AD (based on Bruno et al., to be submitted, see also Table 1). Note that in AD the failure of NGF maturation leads to increased proNGF levels and the activation of MMP-9, which exacerbates degradation of NGF (double trophic factor jeopardy). This situation will deprive forebrain cholinergic neurons of trophic support, and the diminished release of acetylcholine would favor the switch towards an amyloidogenic processing of APP

We also propose that the levels and activity of this NGF maturation/degradation cascade will ultimately play a key role in maintaining the basal forebrain cholinergic phenotype. The uninterrupted supply of NGF should maintain the neuronal phenotype of forebrain cholinergic neurons. Failure of this system should provoke cholinergic atrophy while the preservation of these neurons, in the context of Alzheimer’s disease, would favor a non-amyloidogenic pathway as it has been most elegantly demonstrated by Nitsch and collaborators that the activation of M1 and M3 receptors favors the alpha-secretase type of APP cleavage [17, 18]. Our investigations have shown that the above outlined pathway is operative in vivo conditions. So far, we have intervened at two levels of the proposed cascade. Firstly, we blocked plasmin formation by inhibiting tPA action by infusing the tPA endogenous inhibitor and secondly, by inhibiting the activated MMP-9 by infusing the broad-spectrum MMP inhibitor, GM6001. These experiments confirmed the in vivo validity of the biochemical pathway. Thus, we have found that the continuous, unilateral infusion of the endogenous tPA inhibitor neuroserpin into the cerebral cortex of young rats provoked a several fold increment in proNGF tissue levels, when compared to the contralateral, vehicle-injected side (see Fig. 2A). In these experiments, no change was observed in unrelated, constitutive, proteins. In contrast, the unilateral infusion of the MMP-9 inhibitor, GM6001 in the cerebral cortex of young rats for 72 h, caused a dramatic rise of endogenous mNGF content when compared to values from the contralateral side that received the GM negative control (Fig. 2B).
Fig. 2

The cortical proNGF/matureNGF ratio is changed by the application of neuroserpin or MMP-9 inhibitors (A) Increased amount of cortical proNGF in neuroserpin-treated animals (Mean ± SEM, P < 0.001, t-test). (B) The inhibitor of matrix metalloproteinase GM6001 significantly increased the cortical mNGF (P < 0.001) and decreased proNGF (P < 0.01) when compared with the GM6001 negative or saline control-treated (Mean ± SEM). The levels of neuropsin, a serine protease secretory protein present in pyramidal neurons, and of β-tubulin were not altered in these experiments [16]

Is this newly described mechanism compromised in Alzheimer’s disease? If so, such an event would easily explain the cholinergic vulnerability in AD. We have indeed observed a marked dysregulation of this NGF metabolic pathway in the AD cerebral cortex [19]. In brief, we found a failure in the conversion of proNGF to NGF (lower plasminogen/plasmin activity) which is exacerbated by an increased NGF degradation resulting form a rise in MMP-9 activity. Preliminary animal experimental data would also indicate that a similar dysregulation can be provoked by the injection of Aβ peptides in the hippocampus. In consequence, we hypothesized that pathological alterations of this metabolic cascade in the CNS might be the ultimate cause of the vulnerability of the NGF-dependent forebrain cholinergic neurons observed in AD. In support of this hypothesis is the finding of alterations in the tPA-proteolytic cascade generated by the Aß burden in AD. This situation results in a diminished production of mNGF from the proNGF processing, favoring a non-functional CNS accumulation of proNGF, in line with Fahnestock’s prior observations [13]. In the AD pathology, we additionally observed a remarkable increase in the activity of the NGF-degrading MMP-9 enzyme. Table 1 summarizes our unpublished observations in the human brain [19]. In such a scenario the NGF supply in AD would experience a situation of double jeopardy i.e., diminished production and increase degradation. The resulting failure of NGF trophic stimulation would lead to the progressive atrophy of the basal forebrain cholinergic system and the consequent cholinergic contribution to the AD-related learning and memory decline.
Table 1

(A) Human middle frontal gyrus samples (B) Relative protein levels in AD versus control samples

Panel A





Neurophatological diagnosis




F and M





F and M


Panel B








Alzheimer’s versus control




↑ ↓


F: female; M: Male; NCI (non cognitive impaiment); AD (Alzheimer’s disease) (Panel A)

NSD: non significant differences (Panel B)

In conclusion, we are proposing that the well established vulnerability of NGF-dependent forebrain cholinergic neurons in AD is caused by a profound dismetabolism of the complex protease cascade which is responsible of the maturation and degradation of NGF in the extracellular space. Both animal and human brain data support this hypothesis. We further proposed that the accumulation of Aβ peptides provokes this metabolic dysregulation via mechanisms under investigation. Finally, such a situation would create a “feed-forward” pathological cycle in which the progressive accumulation of Aβ will disregulate the NGF metabolic cascade provoking cholinergic atrophy, which, in its turn, will favor an amyloidogenic metabolism of APP escalating the NGF compromise.


This work was supported by a grant from the Canadian Institutes of Health Research (MOP 62735) and a grant from the US Alzheimer’s Association (IIRG-06-25861). Dr. Claudio Cuello holds a Charles E. Frosst Merck Research Chair in Pharmacology at McGill University.

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© Springer Science+Business Media, LLC 2007