the basal forebrain cholinergic neurons ( bfcn ) provide the major cholinergic innervation to the hippocampus and neocortex , playing a role in cognition and attention behaviors through the release of the neurotransmitter acetylcholine .
bfcn are located in the medial septum , diagonal band of broca , nucleus basalis of meynert and striatum .
they are found to be massively degenerated in late stages of sporadic alzheimer 's disease ( ad ) , one of the most diffuse and lethal disease of the elderly .
however , in contrast with the so called  cholinergic hypothesis  , ad can not be considered as a generalized brain cholinergic disease ( mesulam , 2004 ) .
indeed , the cholinergic system undergoes only a mild reduction of synaptic density and a partial atrophy at the ad onset , while frank bfcn degeneration and death require more than a decade to appear ( mesulam , 2004 ) .
functional bfcn synapses relay on continuous and activity - dependent release of nerve growth factor ( ngf ) by cortical and hippocampal neurons ( iulita and cuello , 2014 ) .
ngf binds to two classes of cell surface receptors localized at the bfcn terminals : the specific ngf receptor tyrosine kinase a ( trka ) and the common neurotrophic receptor p75 . the ngf signal is retrogradely conveyed from axons and dendrites toward the nucleus of bfcn , where it modulates cholinergic gene expression .
the requirement of an active ngf / trka pathway in forebrain - related cognition is confirmed by the positive correlation between trka levels and mini - mental status examination scores . as mentioned above ,
a number of experimental results prompt the perturbation of the ngf pathway as an early event in ad pathology .
in fact , alterations of the ngf / trka signaling system correlate well , and even more robustly than the amyloid load , with cognitive deficits in mci and in its progression toward ad .
moreover , single bfcn expression analysis indicates that trka mrna is reduced in mci , and suggests that decreased neurotrophin responsiveness may be an early ad biomarker ( mufson et al . , 2012 ) .
since the pro - apoptotic ngf precursor ( prongf ) increases while trka levels diminish in the ad forebrain , it is conceivable that degenerative pathways may override ngf - trka survival signals during pre - sympthomatic ad ( mufson et al . , 2012 ) .
further , experimental findings from animal and cellular models indicate that the impairment of the ngf signaling system may be a critical event in the manifestation of this pathology . accordingly , in vitro ngf deprivation in pc12-derived and primary hippocampal neurons
alzheimer 's like molecular syndrome  with both amyloid and tau accumulation ( calissano et al . , 2010 ) .
moreover , antibody - mediated neutralization of ngf promotes the appearance of histopathological signs typical of ad , including amyloid generation and neuronal deficits in the ad11 mouse model ( ruberti et al . , 2000 ) .
thus , it would seem reasonable to antagonize basal forebrain dysfunctions in ad by exogenous ngf administration . in line with this , intracerebral ngf supply has been found beneficial to cholinergic neurons and related behavior in rodents . in particular
, nasal administration of ngf modulates secretases levels and reduces amyloid burden in app / ps1 transgenic mice ( yang et al . , 2014 ) . of note
, ngf gene therapy has been attempted in a phase 1 clinical trial , which showed high tolerability and lack of side effects in ad patients . also , the results of a 10 years long study confirmed safety and efficacy of the ngf therapy , and reported long lasting brain responsivity to ngf in terms of activation of functional markers , hypertrophy , and axonal sprouting ( tuszinsky et al . , 2015 ) . as less invasive alternatives to intracerebral stereotaxic ngf delivery
, the ngf administration via the ocular or nasal routes has also been performed in rodents and promoted cholinergic system neuroprotection . all together , these findings underline the importance of a proper homeostatic regulation of the neurotrophic pathway in the early phase ad and pinpoint the feasibility of ngf therapy , claiming the need for efficient , safe and long - lasting therapeutic approaches for the ngf treatment of ad .
activity - dependent release of ngf from cortical and hippocampal neurons has been demonstrated to sustain the cholinergic tone on bfcn target neurons via the muscarinic receptors 1 ( m1 ) .
ngf has been thought to maintain neuronal homeostasis and forebrain - related cognition mainly through this interplay .
in fact , m1 activation induces the physiological cleavage of app to generate soluble app ( sapp ) , which is neuroprotective per se , and it is also a potent inhibitor of the enzyme responsible for the amyloidogenic app cleavage , the beta secretase 1 ( bace1 ) . whether ngf directly regulates app metabolism in bfcn
indeed , we recently demonstrated that the ngf signaling pathway is able to modulate app processing in bfcn both in vitro and in vivo ( triaca et al . , 2016 ) .
we showed that stimulation of primary cholinergic septal neurons with ngf promotes binding of the ngf receptor trka with app and the preferential app trafficking to the golgi compartment , where app binding to and cleavage by bace1 is hampered . as a result
, the levels of bace - generated app fragments , like soluble app  ( sapp ) , c - terminal fragment  ( ctf ) and beta amyloid ( 142 ) , are strongly reduced . in particular , we observed that binding of trka to app is facilitated by ngf through the reduction of app phosphorylation at the threonine 668 ( t668 ) residue of its cytosolic tail .
in fact , co - localization and co - immunoprecipitation analyses showed that trka fails to bind app molecules phosphorylated at t668 ( app ) , suggesting that app phosphorylation prevents app binding to trka as already observed for another app interactor , namely fe65 .
app phosphorylation at t668 is a post - translational modification known to facilitate app cleavage by bace1 and amyloid generation , and it was proposed as target for ad therapy ( lee et al . , 2003 ) .
thus , ngf exerts its anti - amyloidogenic action by lowering the fraction of app molecules , favoring trka - app interaction and the subsequent app processing along the anti - amyloidogenic route .
the early downstream molecular players involved in the control of app metabolism have also been investigated . upon ngf binding , trka activation promotes the phosphorylation of the isoform c of the sh2 containing sequence ( shc ) , the early trka adaptor expressed in adult bfcn .
afterward , shcc activation inhibits the p54 isoform of the c - jun n - terminal kinase p54 ( jnk ) , a well known ser / thr app kinase , thus reducing app levels , and promoting app - trka binding ( figure 1 ) .
a schematic model illustrating amyloid precursor protein ( app ) metabolism control by the nerve growth factor / tyrosine kinasea ngf / trka signaling system in healthy basal forebrain cholinergic neurons ( bfcn ) ( anti - amyloidogenic route , left ) , and the consequences of its perturbation in alzheimer 's disease ( amyloidogenic route , right ) . anti - amyloidogenic route .
, trka trans - phosphorylation takes place , inducing trka phosphorylation at the tyrosine 490 ( y490 ) residue and trka docking of the signaling adaptor sh2 containing sequence c ( shcc ) .
once activated , shcc inhibits c - jun n - terminal kinase ( jnk ) , a ser / thr app kinase thus hindering the phosphorylation of app at the threonine residue 668 ( t668 ) . since trka is able to bind only app molecules not phosphorylated at t668 , the reduction of app levels induced by ngf promotes 1 ) app - trka binding , 2 ) subsequent trka - mediated trafficking of app to the golgi compartment and to the plasmamembrane , and 3 ) preferential cleavage of app by the neuronal alpha secretases 1017 ( adam1017 ) through the physiological pathway .
reduced availability of mature ngf , and/or expression levels of trka affect app metabolism in bfcn .
in fact , disturbances in the anti - amyloidogenic ngf / trka - shcc signaling pathway grant the activation of jnk by pro - apoptotic signals , resulting in augmented app levels and disruption of app interaction with trka in favor of beta secretase 1 ( bace1 ) binding and cleavage of app along the amyloidogenic pathway .
, 2016 . the app - trka interaction seems to have a pathological significance in ad .
in fact , app - trka interaction is specifically lost in ad affected tissues , like the hippocampus , while it seems to be preserved in the ad cerebellum , as well as in the hippocampus of patients affected by other neurodegenerative diseases , like huntington 's disease .
a deeper analysis of the app - trka interaction in bfcn in vitro and in vivo by proximity ligation assay ( pla ) and bimolecular fluorescence complementation ( bifc ) is currently ongoing in our group .
altogether , these findings suggest that the ngf system maintains amyloid levels within the physiological range in healthy bfcn by modulating app processing by bace1 .
based on reduced trka and/or ngf levels observed in mci and early ad , it is tempting to speculate that disturbances in attention and cognition may result from the perturbation of the ngf - trka - shcc pathway in bfcn , inducing and/or contributing to synaptic deficits of their hippocampal and cortical target neurons .
it is well known that bfcn are more vulnerable to ad , as compared to those located in the cerebellum .
higher forebrain susceptibility to intraneuronal amyloid accumulation has been suggested to account for this difference .
intraneuronal amyloid accumulation has been extensively demonstrated to occur during brain ageing and in ad pathology in the bfcn of mice , monkeys and humans by the geula lab ( baker - nigh et al . , 2015 ) .
a substantial increase of intraneuronal amyloid long before plaques formation has also been reported in 3xfad transgenic mice ( la ferla et al . , 2007 ) . while intraneuronal amyloid is neuroprotective against oxidative stress at physiological levels ( picomolar ) , higher concentrations
therefore , intraneuronal amyloid accumulation has been prospected as a good predictor of synaptic and neuronal loss ( bayer and wirths , 2010 ) . as elegantly demonstrated by laferla et al .
( 2007 ) , the newly generated amyloid first appears inside neurons and afterwards outside the cells , suggesting that intraneuronally generated amyloid can be released into the extracellular space causing plaque deposition in 3xfad transgenic mice .
novel findings from clinical examinations , amyloid imaging , and functional mri provide evidence that not only neocortical regions , but also subcortical areas of the basal forebrain ( e.g. , striatum ) show amyloid accumulation and neurodegeneration at the pre - symptomatic ad stage . accordingly
, early ad pathology is characterized by signs and symptoms of dysfunctional subcortical circuits ( shinohara et al . ,
2014 ) . in line with this , the study of amyloid accumulation in the bfcn of mouse models lacking the mature ngf signaling will be instrumental in the pathological and molecular profiling of the ad onset .
based on the relevance of ngf signaling in the physiological control of app processing in the basal forebrain , it can be hypothesized that lack of neurotrophic support may boost amyloid generation and intracellular accumulation in bfcn , thus promoting the initial synaptic disturbances seen in mci and early ad . here , we prospect that upon ngf withdrawal cholinergic neurons may primarily contribute to ad pathology and affect target neurons in the cortex and hippocampus by generating and releasing amyloid , possibly through the exosomal and/or synaptic routes . on the other hand ,
the newly generated amyloid is able to inhibit the endocytosis of the ngf / trka complex at the cholinergic terminals , in a negative feedback loop which settles the ad onset ( kim et al . , 2016 ; xu et al . , 2016
once age - related events ( oxidative stress , astrogliosis , reduced amyloid clearance ) occur , they compromise the brain buffering capacity and determine the overt neuronal loss of bfcn and their targets typical of late stage ad
. the fine analysis of the spatio - temporal sequence of amyloid appearance in the ad brain will hopefully provide important insights into the pathological drivers of this devastating neurodegenerative disease of the elderly , paving the way for novel targeted approaches in ad therapy .
 the current idea behind brain pathology is that disease is initiated by mild disturbances of common physiological processes . 
 overtime , the disruption of the neuronal homeostasis will determine irreversible degeneration and neuronal apoptosis . 
 this could be also true in the case of nerve growth factor ( ngf ) alterations in sporadic alzheimer 's disease ( ad ) , an age - related pathology characterized by cholinergic loss , amyloid plaques and neurofibrillary tangles . 
 in fact , the pathway activated by ngf , a key neurotrophin for the metabolism of basal forebrain cholinergic neurons ( bfcn ) , is one of the first homeostatic systems affected in prodromal ad . 
 ngf signaling dysfunctions have been thought for decades to occur in ad late stages , as a mere consequence of amyloid - driven disruption of the retrograde axonal transport of neurotrophins to bfcn . nowadays , a wealth of knowledge is potentially opening a new scenario : ngf signaling impairment occurs at the onset of ad and correlates better than amyloid load with cognitive decline . 
 the recent acceleration in the characterization of anatomical , functional and molecular profiles of early ad is aimed at maximizing the efficacy of existing treatments and setting novel therapies . 
 accordingly , the elucidation of the molecular events underlying app metabolism regulation by the ngf pathway in the septo - hippocampal system is crucial for the identification of new target molecules to slow and eventually halt mild cognitive impairment ( mci ) and its progression toward ad . 
NGF Pathway Dysfunctions in Mild Cognitive Impairment and AD Neurodegeneration: Lessons from Human Studies and Animal Models
Signaling and Molecular Mechanisms Responsible for the Regulation of APP Processing by the NGF/TrkA Pathway in the Basal Forebrain
Intraneuronal Amyloid Accumulation in BFCN during Mammalian Brain Ageing and Neurodegeneration: is Disrupted NGF Signaling the Culprit?
Perspective