Drug target identification at the crossroad of neuronal apoptosis and survival

ABSTRACT

Introduction: Inappropriate activation of apoptosis may contribute to neurodegeneration, a multifaceted process that results in various chronic disorders, including Alzheimer’s and Parkinson’s diseases. Several in vitro and in vivo studies demonstrated that neuronal apoptosis is a multi-pathway cell-death program that requires RNA synthesis. Thus, transcriptionally activated genes whose products induce cell death can be triggered by different stimuli and antagonized by neurotrophic factors. Systems biology is now unveiling the series of intracellular signaling pathways and key drug targets at the intersection of neuronal apoptosis and survival.

Areas covered: This review introduces a genomic approach that can be used to elucidate the systems biology of neuronal apoptosis and survival, and to rationally select drug targets, no longer oriented to emulate the action of growth factors at the membrane receptor level, but rather to modulate their downstream signals.

Expert opinion: The advent of genomics is offering an unprecedented opportunity to explore how the delicate balance between apoptosis and survival-inducing signals triggers a transcriptional program. Characterization of this program can be useful to identify potential pharmacological targets for existing drugs. Such knowledge might pave the way towards an innovative pharmacology.

KEYWORDS:

• Apoptosis
• cerebellar granule neurons
• drug target
• insulin-like growth factor-1
• pituitary adenylyl cyclase-activating polypeptide
• systems biology

1. Introduction

Selective neuronal loss, accompanied by specific protein aggregation, is the histopathological hallmark of neurodegenerative diseases. Although the abnormal proteins responsible for each disease are different in structure and function, all neurodegenerative disorders share the common process of protein misfolding and aggregation. These aggregates directly and indirectly attack cellular components, leading to neuronal death [1]. For many years, two cell death pathways, namely, apoptosis and necrosis, have been considered as the main forms of cell death.

Apoptosis is a genetically controlled program of cell death by which a cell regulates its natural self-destruction. This intrinsic suicide process involves a well-choreographed sequence of biochemical and morphological events, including fragmentation of nuclear DNA, breakdown of cellular cytoskeleton, cell shrinkage, pyknosis, and blebbing of the plasma membrane, which breaks up into membrane enclosed particles, termed apoptotic bodies [2].

Necrosis is not a regulated form of cell death and is characterized by plasma membrane disruption, organelle and cytoplasm swelling, and release of intracellular contents onto extracellular compartment. Excitotoxicity is a characteristic of the necrotic death and is due to a fast release of Ca²⁺ from intracellular pools, activating calpains that cleave cytoskeleton proteins [3].

Even though necrosis is characterized by the lack of a clear molecular pathway, a necrosis-like process, partially programmed, seems also to exist. This sort of programmed necrosis, known as necroptosis, has recently been recognized and has the characteristics of apoptosis and necrosis, being a caspase-independent cell death, driven by defined molecular pathways [4]. Necroptosis has been identified as a strong trigger of innate and adaptive immune responses, evolved as a line of defense against intracellular infection [5].

While both necrosis and necroptosis prevalently occur in acute brain injuries, such as ischemic damage [6], there is little doubt that apoptosis plays a major role in neurodegenerative diseases [7].

Apoptosis is intimately linked to sculpting the correct architecture and size of neuronal and glial populations of the nervous system [8]. Following the developmental window, aberrant neuronal apoptosis is thought to contribute to the development of a series of neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases [9]. Owing to this leading role in neurodegenerative disorders, the major interest has traditionally been directed to elucidating the molecular mechanisms underlying neuronal apoptosis and devising strategies aimed at counteracting it.

Neurotrophic factors have been identified as the key factors in antagonizing neuronal apoptosis and promoting life signals in neuronal cells [10,11]. Delivery to the brain of these peptide molecules, however, is normally hindered by the blood–brain barrier (BBB), which limits the therapeutic utility of their exogenous administration [12]. To circumvent this problem, there has been increasing focus on the development of pharmacological strategies no longer oriented to emulate the action of growth factors at the membrane receptor level, but rather to modulate their downstream signals.

The advent of high-throughput technologies has dramatically accelerated efforts in the field of systems biology to understand the transcriptional mechanisms underlying the action of neurotrophic factors under a variety of experimental physiological and pathological conditions [13–16]. Such knowledge is vital in identifying drug targets downstream of the neurotrophic factor receptors and paving the way toward the future aim of transforming genes into pharmaceutical compounds with neurotrophic activity that can interfere with the apoptosis machinery and be effective against untreated neurodegenerative diseases.

This review will specifically focus on recent advancements in the systems biology of neuronal apoptosis and survival, demonstrating the utility of a genomic-based approach to explore pathways and potential drug targets at the crossroad of neuronal apoptosis and neurotrophic factor rescue.

2. Genomic approach

Over the past 20 years, global analysis of mRNA expression has emerged as a powerful strategy for biological discovery. Using the power of parallel processing, robotics, and computer-based informatics, a number of high-throughput genomic technologies have been devised. These include whole-genome DNA microarrays and, most recently, RNA-sequencing (RNA-seq), each one having its own advantages and disadvantages [17]. Before the advent of these two technologies, the majority of studies focused on individual genes or altered pathways and their collective behavior was therefore unknown. Microarray technology was the first to make possible whole-genome transcriptional analysis. Although the cost of microarray analysis is still lower than sequencing, RNA-seq is gaining more users among researchers. Among its major advantages is the ability to have direct access to the RNA sequence, the exon/exon boundaries and, therefore, produce a better picture of alternative splicing isoforms. Following both microarray and RNA-seq analysis (Figure 1), deregulated genes and enriched pathways can be identified by means of a variety of bioinformatic and statistical tools that, once applied to large-scale genomic and gene expression data, help in interpreting their biological role [18,19].

Figure 1. Genomic approach for drug discovery in CGNs. A drug target discovery pipeline starting from microarray and RNA-seq analysis moves through drug targets identification and ends with their validation in in vitro models.

Microarray technology has been the most used approach to assess the systems biology of neuronal apoptosis and survival [20]. In particular, the advent of microarrays has profoundly contributed to elucidating the existence of transcriptional pathways and a wider spectrum of pro- and antiapoptotic mediators then were originally believed to exist. Such knowledge has proved extremely useful in identifying drug targets downstream of the neurotrophic factor receptors and is paving the way toward the transformation of genes into pharmaceutical compounds with neurotrophic activity that can interfere with the apoptosis machinery and be effective against untreated neurodegenerative diseases.

The following part of this review will show some examples of how the genomic approach has been applied to dissecting the gene-based mechanisms underlying neuronal apoptosis and survival, describing the most important studies that have contributed to understanding them.

3. Application of the genomic approach to the study of neuronal apoptosis and survival

Since transcription has emerged as an obligate component of neuronal programmed cell death (PCD) and survival, microarray technology represents an excellent tool to fully understand transcriptional changes occurring during these processes. With the advent of DNA microarray, it has been possible to monitor the global expression changes occurring in numerous in vitro and in vivo neuronal paradigms.

The first paper in the literature that studied the gene expression component of neuronal apoptosis was that of Chiang et al. [21]. By using a brain-biased array, this study profiled the gene expression changes of cerebellar granule neurons (CGNs) induced by different apoptotic signals, including potassium and serum withdrawal (KS-W) [21]. Important progress in understanding the apoptotic program elicited by KCl deprivation derived from genome scale studies based on oligonucleotide microarrays, which identified a great number of temporally coupled genes that delineate functional pathways mobilized during neuronal PCD [14,18]. The limitations of the first studies in encompassing the entire set of deregulated genes in neuronal apoptosis were overcome with the advent of whole-genome microarrays, which allowed the whole apoptotic program to be analyzed [19]. A comparative study between different in vitro paradigms of apoptotic neuronal death has subsequently indicated the existence of common regulatory mechanisms underlying neuronal cell death [13]. Comparison of other gene expression data arising from postmortem brain tissues affected by neurodegenerative disorders has provided the first comprehensive view of the molecular alterations that seem to contribute to human neuropathogenesis [22] and offered an outstanding opportunity not only to look into exclusive disease-specific changes, but also more importantly to look for potential common molecular pathogenic mechanisms [23].

Most of the current knowledge about the transcriptional changes occurring during neuronal death has been obtained using microarray technology. Microarray analysis, however, is capable of measuring only the status of known transcripts. In addition, expression of low-abundance mRNAs is not detected by this hybridization-based approach. Using RNA-seq, a recent study enabled additional genes, deregulated during the process of neuronal death, to be identified [24].

DNA microarray has proved useful for screening cellular targets that govern apoptosis [18,25]. Our most recent studies, in particular, were the first to extend the analysis to survival and led to the identification of key drug targets deregulated in both apoptosis and growth factor rescue [11,15,26]. The following paragraphs will describe how this kind of approach was first utilized to characterize the transcriptional program at the intersection of apoptosis and survival of CGNs and to identify new potential drug targets.

4. Transcriptional programs of neuronal apoptosis and survival in CGNs

One basic feature of neuronal apoptosis in vivo is its sporadic and asynchronous nature. In such a condition, neurons undergo apoptosis at very different times and this may be attributed to a variety of factors, such as date of generation, local environments, and input received from other cells [2]. On the contrary, a synchronous population of neurons can undergo apoptosis in in vitro systems, where most of these variables can be controlled and studied more effectively. In vitro models, therefore, represent an important step in the search for potential drug targets, as they substantially reduce the complexity of in vivo models.

CGNs are the most abundant neuronal type in the mammalian brain and represent the model most commonly used either in vivo or in vitro to study neuronal apoptosis and survival [27]. Although CGNs do not recapitulate all features of human neuropathology, they have been extensively used to evaluate the pathogenic mechanisms underlying neurodegeneration and to validate potential therapeutic compounds.

During early stages of postnatal development, it is assumed that apoptosis of granule cells reflects the failure of these neurons to obtain adequate amounts of specific neurotrophic factors [28]. In the adult, mossy fiber axotomy is followed by apoptosis of CGNs, underlining the crucial role of afferent inputs for the survival of these cells [27]. In vitro, CGNs undergo rapid apoptotic cell death within 24 h after removal of serum and lowering of extracellular potassium from 25 to 5 mM [27,29]. Apoptosis of CGNs requires protein transcription and synthesis, becoming irreversible after the first 6 h following its induction. Before this ‘commitment point,’ CGNs can be rescued by the activation of specific signal transduction pathways or by the treatment with specific neurotrophic factors [27,29].

Two of the most effective growth factors capable of preventing apoptosis of CGNs are insulin-like growth factor-1 (Igf1) [29] and pituitary adenylyl cyclase-activating polypeptide (Pacap) [30]. Although the survival effects of these neurotrophic factors are mediated by different receptors and intracellular second messengers [31,32], they are propagated by common transcriptional cascades and show striking similarities [33] (Figures 2 and 3). Indeed, not only a majority of differentially expressed genes, defined as ‘survival related genes’ (SRGs), are common to Igf1 and Pacap (Figure 2), but also the Pearson correlation coefficient between Igf1 and Pacap for all SRGs is 0.97 [33]. Taken together, these data support the existence of a preserved transcriptional program underlying neuronal survival. Figure 3 shows a comprehensive picture of differently expressed genes and enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways associated with the rescue of CGNs following treatments with Igf1 and Pacap. Several SRGs encode drug targets for existing drugs. In the next section, these targets and their related pharmacological compounds will be described.

Figure 2. Genes differentially expressed in CGNs after induction of apoptosis and rescue by Igf1 and Pacap. (a) Venn Diagram. Figure shows the 1212 genes differentially expressed in apoptotic CGNs (K5 vs K25) defined as ‘Apoptotic related genes’ (ARGs), and the 1535 genes differentially expressed in rescued CGNs (K5 + Igf1 vs K5; K5 + Pacap vs K5) defined as ‘Survival related genes’ (SRGs). (b) Hierarchical cluster. A hierarchical clustering algorithm was used to order the differential expressed genes (ARGs and SRGs) in a dendrogram, in which the pattern and length of the branches reflects the relatedness of the expression levels in the different experimental conditions. Data are presented in a matrix format: each row represents a single gene and each column an experimental condition. As shown in the color bar, the red color indicates highly expressed genes, the blue color down-regulation genes.

Figure 3. A comprehensive picture showing deregulated genes and pathways linked to Igf1 and Pacap rescue of apoptotic CGNs. This figure, illustrates the gene expression profiles and the 14 KEGG pathways that are mainly involved in Igf1 and Pacap neuroprotection of apoptotic CGNs. Each encoded protein is labeled with two thermometers that indicate gene expression ratios under the following experimental conditions: K5+Igf1/K5 (first thermometer) and K5+Pacap/K5 (second thermometer). More specifically, downward thermometers have a blue color and reflect down-regulated expression, whereas upward thermometers have a red color and reflect up-regulated expression following Igf1 or Pacap treatment. Several of the deregulated genes encode drug targets for known pharmacological compounds (indicated by ‘R’ in a small green rhombus). Some of the drug targets discussed in the manuscript are highlighted by a gray circle.

5. Potential drug targets at the crossroad of neuronal apoptosis and survival

As mentioned above, transcriptional analysis of CGNs unveiled a large number of genes and pathways that are commonly deregulated during apoptosis and its rescue by Igf1 and Pacap [11]. Several of these genes encode known drug targets, which include ligands, G-protein-coupled receptors, ion channels, enzymes. and transcription factors (Figure 3 and Table 1). A comprehensive list of potential drug targets with their pharmacological compounds is shown in supplementary Table S1.

Table 1. Potential drug targets at the intersection of apoptosis and survival following treatment with Igf1 and Pacap.

Gene symbol	Target	Gene symbol	Target
Ntrk1	TrkA	Nmnat2	NMNA2
Odc1	DCOR	Fxyd2	FXYD2
Ryr1	Ryanodine receptor 1	Adamts2	ADAM-TS2
Pdgfb	PDGF-B	Mvd	MVD
Cacna1s	CACNA1S	Adam1a	ADAM1A
Grin3b	NR3B	Zfp612	ZNF23
Pdk1	PDK1	Aco1	IRP1
Adora1	Adenosine A1 receptor	Il2rg	IL-2R gamma chain
Src	c-Src	Limk2	LIMK2
Cacna1d	CACNA1D	Gpld1	PHL1
Cdh2	N-cadherin	Nhlh2	NHLH2
Tgm2	TGM2	St3gal5	GM3 synthase
Atp1a1	ATP1A1	Cldn1	Claudin-1
Casp7	Caspase-7	Kcnq2	KCNQ2
Nr1h4	FXR	Asah1	ASAH
Ret	RET	Ccl12	CCL2
Crhr1	CRHR1	Slc25a21	SLC25A21
Fntb	FTase-beta	Cyp2b2	CYP2B6
Car4	Carbonic anhydrase IV	Paqr7	PAQR7
Nae1	APP-BP1	Cubn	Cubilin
Atp12a	ATP12A	Pthlh	PTHrP
Asah1	ASAH	Bdkrb1	BDKRB1
Xdh	Xanthine oxidase	Krt1	Keratin 1
Gart	PUR2	Ccl2	CCL13
KV1.6	KV1.6	Tnnc1	Troponin C, cardiac
Csf1r	M-CSF receptor	Arfgap3	ARFGAP3
Mmp16	MMP-16	Cebpb	C/EBPbeta
Txnrd1	TXNRD1	Gja8	Connexin 50
Hdac7	HDAC7	Hspa5	GRP78
Gabrb2	GABA-A receptor beta-2 subunit	Trib3	NIPK
Tuba4a	Tubulin alpha-4A	Shc1	Shc
Crtc2	TORC2	Zdhhc4	ZDHHC4
Slc6a2	NET	Dio3	DIO3
Kcnk2	KCNK2	P2rx6	P2X6
Kcnc1	Kv3.1	Cybb	gp91-phox
Psenen	Pen-2	Fstl3	FSRP
Pim2	Pim-2	Gadd45a	GADD45 alpha
Hdac4	HDAC4	Zfp74	ZNF569
Pdpk1	PDK (PDPK1)	Cyp2d4	CYP2D6
Top2a	TOP2 alpha	Npbwr1	GPR7
Pde4b	PDE4B	Cdh6	K-cadherin (CDH6)
Cdk4	CDK4	Slc6a6	SLC6A6
Timp2	TIMP2	Asl	ARLY
Mcl1	Mcl-1	Zfp148	ZNF148
Flt3	FLT3	Ptpn9	PTP-MEG2
Htr3b	HTR3B	Slco4a1	OATP-E
Slc22a13	SLC22A13	Npr3	NPR3
Zdhhc8	ZNF378	Grk5	GRK5
Plg	Plasminogen	Cyp3a23/3a1	CYP3A1
Tnc	Tenascin-C	Anxa6	Annexin VI
Hsd3b5	HSD3B5

In the following paragraphs, some of these drug targets will be discussed by grouping them into three main functional clusters: cellular processes, signal transduction, and metabolism. Particular attention will be given to those drug targets that have been already implicated in neurodegenerative diseases and cancer, which may be influenced by common signaling pathways [34].

5.1. Cellular processes

Transcriptional signaling pathways underlying rescue of apoptosis in CGNs by Igf1 and Pacap include genes functionally involved in specific cellular processes: (1) antigen processing and presentation, (2) cytoskeleton remodeling, and (3) cell death. Among potential deregulated therapeutic targets, Hspa5 (i), Limk2 (ii), Mcl1 (iii), and Gja8 were all found to be upregulated by Igf1 and Pacap. Inhibition of Gja8 by 18alpha-glycyrrhetinic acid [35] and activation of Hspa5 by salubrinal, 2DG, and NE-100 [36,37] have already been shown to support cell survival. Similarly, treatment with LIMKi and A-1210477, two selective inhibitors of Limk2 and Mcl1, respectively, prevents apoptosis in cancer [38,39].

5.2. Metabolism

Several enzymes linked to a number of metabolic pathways are deregulated during neuronal apoptosis and survival in vitro. Their additional involvement in neurological disorders has stimulated great interest in their pharmacological modulation. Below, we will discuss some of these potential metabolic drug targets.

5.2.1. Enzymes involved in extracellular matrix remodeling

Mmp16, Adamts2, and Adam1a are members of the matrix metalloproteinase (MMP), a disintegrin, and metalloproteinase with thrombospondin motifs (ADAMTS) and metalloproteinase-disintegrins (ADAMs) family of proteins, participating in the remodeling of the extracellular matrix. Although they are emerging as regulators of nervous system development and adult brain functions, their role in cell death remains to be elucidated [40,41]. Our in vitro data, however, show an increased expression of Mmp16 and Adam1a and the decrease of Adamts2 during CGNs rescue [15]. To clarify the role of these enzymes, additional studies should be performed to investigate the neuroprotective effects of metalloproteinase inhibitors, like BB-94 [42] and marimastat [43], or activators like losartan [44].

5.2.2. Enzymes in anabolic and catabolic chemical reactions

Aco1, Asl, Dio3, Gpld1, Hsd3b5, Odc1, and Nmnat2 are enzymes involved in a series of catabolic reactions that were upregulated in rescued CGNs, while Asah1 was downregulated by Igf1 and Pacap. With the exception of Aco1/Irp1, which is already a known target of neuroprotective drugs like tempol or iron chelators (R-apomorphine) [45,46], the functional effect of pharmacological modulation of the other enzymes remains to be clarified. Some studies have focused on the inactivation of these enzymes in a range of malignancies. Drugs like pegylated arginine deiminase/ADI-PEG20 (Polaris Group) (Als inhibitor) [47], xanthohumol (Dio3 inhibitor) [48], 1,10-phenanthroline (Gpld1 inhibitor) [49], compounds 54, 66, and 67 (inhibitors of Hsd3b5) [50], and alpha-difluoromethylornithine (Odc1 inhibitor) [51] have already been reported to act as sensitizing anticancer agents.

Deregulation of Asah1, an acid ceramidase, and Nmnat2, a nicotinamide mononucleotide adenylyltransferase (NMNAT) enzyme, has been involved in a variety of neurodiseases and/or cancers [52,53]. Recent studies have reported that Asah1 inhibitors, such as B-13, Ceranib 2, or LCL 204, slow the growth of cancer cells, alone or in combination with other established, antioncogenic treatments [54,55]. Although there is no evidence of selective Nmnat2 drugs, palmostatin B is able to boost Nmnat2 palmitoylation that is important for promoting survival of axons and preventing the onset of neurodegenerative disorders [56,57].

5.2.3. Cytochrome enzymes

Transcriptomic analysis of CGNs undergoing apoptosis or growth factor rescue unveiled the deregulated expression of a number of cytochrome b and P450 enzymes, which are some of the most versatile redox proteins known. Among these, Cyp3a23/3a1 and Cyp2d4 were overexpressed, while and Cybb and Cyp2b2 were downregulated in rescued CGNs.

Cyp3a23/3a1 is well recognized for its role in the metabolism of numerous drugs and xenobiotic compounds in several tissue types. Several agents, such as fluconazole [58], myclobutanil, triadimefon [59,60], beta-naphthoflavone, phenobarbital [61], and triadimefon [59], can induce the family of CYP3A isoforms in rat, mouse, and human in a dose-related manner. In contrast, troleandomycin and apatinib are able to selectively inhibit Cyp3a23/3a1 [62]. Cyp2b2 is known to protect neuronal cells from exogenous neurotoxic compounds (i.e. 7-pentoxyresorufin-O-dealkylase) [63] and Cyp2b2 activators have already been demonstrated to have antioxidant effects in rat liver [64]. Cyp2d4 regulates neuronal survival when pharmacologically inhibited by herba erigerontis and fluoxetine drugs [65,66].

5.2.4. Palmitoyltransferase pathway

In rescued CGNs, two proteins containing DHHC-type zinc finger domain, Zdhhc4 and Zdhhc8, were found to be downregulated. These are stable scaffolds that have evolved a palmitoyltransferase activity, which plays a critical function throughout the nervous system [67,68]. Zdhhc8, in particular, influences mitochondria-regulated apoptosis when overexpressed [69]. Chemical inhibitors of palmitoylation, such compound V and 2BP [70], may be used to test the direct involvement of these proteins in neuronal apoptosis and survival.

5.3. Signal transduction

In rescued CGNs, deregulated expression of genes encoding proteins of the neuroactive ligand and receptor interaction pathway, ion channels, chemokine, MAPK, TNF, and transcription factors signaling were observed.

5.3.1. Neuroactive ligand and receptor pathway

A number of G-protein-coupled receptors were differently expressed in CGNs rescue. Overexpression of Adora1, Grk5, and Npr3, as well as downregulation of Bdkrb1, Npbwr1, and Htr3b, is only a few examples of G-protein-coupled receptors linked to Igf1 and Pacap neuroprotection. Several studies have assessed that selective Adora1 agonists, CCPA and CPA, induce neuroprotection [71]. These findings imply that the drug manipulation of Adora1 may facilitate the treatment of Parkinson’s and Alzheimer’s diseases [72,73]. Pharmacological modulation of Bdkrb1 by its agonist R838 is considered a target for the treatment of chronic inflammatory diseases such as multiple sclerosis [74]. Grk5 antagonists, such as amlexanox [75], Npbwr1, (5-chloro-4-(4-methoxyphenoxy)-2-(p-tolyl)pyridazin-3(2H)-one) [76], Htr3b, MDL72222, and Y25130 [77], have protective effects against inflammatory and/or stress-induced apoptosis, highlighting the importance of further investigation in neurodiseases where these genes have been involved [78–80]. Finally, induction of Npr3 by its agonist cANF⁴⁻ [23] exerts cytoprotective effects in nonneuronal cells [81].

5.3.2. Ion channels

Within the genes identified in rescued CGNs, several ion channels are differentially expressed. Among these, Kcna6 was found to be downregulated, while Kcnc1 and Kcnq2 were upregulated. Kcnc1 and Kcna6 are potassium channels, highly expressed in rat cerebellum [82], which are effective in restoring axonal conduction in spinal cord injury and multiple sclerosis when antagonized by voltage-gated K⁺ channel blockers like 4-AP-3-MeOH and 4-AP [83]. Over-activation of certain potassium channels, like Kcnq2, has been also associated with the early ionic events during the apoptotic cascade. As reported in previous studies, the potent Kcnq2 inhibitor, XE991, suppresses in vitro neuronal cell death [84]. Thus, further pharmacological modulation of Kcnq2 with selective activators like 5-(2,6-dichloro-5-fluoropyridin-3-yl)-3-phenyl-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one [85] should be tested as an important pharmacological strategy against neurodegeneration.

5.3.3. Chemokine, MAPK, and TNF signaling

Among the chemokine-signaling genes deregulated during CGNs rescue is the overexpressed Ccl2/Mcp1. Accumulating evidence indicates a dual role of Ccl2 in neuronal survival [86,87]. While drugs like bindarit downregulate Ccl2 and protect neurons [88], noradrenaline transporter inhibitors like desipramine elicit neuroprotection by upregulating the expression of Ccl2 [89,90].

Among the potential drug–targets linked to Mapk signaling, we detected the downregulation of Shc1 and the overexpression of Mapk3/Erk1, Map2k3, and Gadd45α in rescued CGNs. Given the significant role Mapk proteins play in cancer development and progression, intense research activities were directed toward pharmacological modulation of Mapk pathway. Potent and selective cell permeable inhibitors of Shc1 (i.e. CGP78850) and Mepk3/Erk1 (i.e. BAY 43-9006 and PD98059) have already been shown to be beneficial in suppression of cancer cell growth [91,92]. Selective inhibitors of Map2k3 have been well characterized by fragment-based drug design and further studies are necessary to validate their potential impact in diseases [93]. Although there is no selective drug for Gadd45, fucoxanthin, docetaxel, peptidylarginine deaminase 4, and histone deacetylase inhibitors are Gadd45 activators that are able to inhibit cancer cell growth [94].

5.3.4. Transcription factor signaling

Among the differentially expressed genes that encode transcription factors involved in CGN rescue are c-Fos, c-Jun, Srf, Nur77, and Myc, which are potential drug targets for several activator and/or inhibitor compounds (Table 1). A number of previous, experimental studies show that the pharmacological targeting of these molecules may favor cell survival [95].

6. Pharmacological modulation of myc, a potential drug target downstream of growth factors

Transcription factors have recently received considerable attention as potential targets for counteracting inappropriate activation of neuronal apoptosis due to their significant role to act downstream of signaling cascades and control global gene expression in response to a specific stimulus. Although this interest has produced encouraging results, much still remains to be investigated, especially concerning the effective targeting of these molecules with therapeutic compounds. Among the groups of differentially expressed genes encoding for transcription factors during CGNs rescue by growth factors, we focused on Myc. This transcription factor is known to play a key role in neuronal apoptosis by regulating the expression of many genes through binding enhancer box sequences (E-boxes) and recruiting histone acetyltransferases. Based on these observations, we decided to investigate the role of Myc in neuronal survival by studying the effects of its inhibitor, 10058-F4. As shown in our previous work [26], treatment with this compound was able to reduce the rescue effect of growth factors. Although preliminary, these experiments support a new pharmacological strategy based on the systems biology of neuronal apoptosis and survival, no longer oriented to emulate the neuroprotective action of growth factors at their membrane receptor level but, rather, to modulate their downstream signals.

7. Conclusion

Although this review demonstrates the utility of DNA microarray technology as a mean for dissecting the multigenic programs at the crossroad of neuronal apoptosis and survival, the presented data represent just a glimpse of their complexity. In light of the recent description of the involvement of necroptosis in neurodegeneration, it is conceivable that the large number of genes and pathways that are commonly deregulated during apoptosis could also influence regulated necroptosis, since they may be both not mutually exclusive but proceed simultaneously. Indeed, rather than a ‘switch’ operating between the two modes of cell death, the final mechanism may depend on levels of the respective signaling and effector proteins. Understanding how the brain is self-programmed to counteract neurodegeneration in a concerted and structured manner, with the interplay of different cell death processes, represents a future key for the development of appropriate and effective therapeutic approaches. Therefore, monitoring of gene expression, in different neuronal types, remains the next challenge and will require further bioinformatic advancements. The comprehensive understanding of the genes or sets of genes that play critical roles in neuronal death and survival will represent the starting point to elaborate new pharmacological therapies against neurodegenerative disorders.

8. Expert opinion

There is a vast amount of evidence indicating that neurotrophic factors are diffusible peptides that play a key role in modulating the development, maintenance, and survival of neurons and neuron-supporting cells. In addition, it is well known that alterations in levels of neurotrophic factors can lead to a dramatic cell loss through a transcription-dependent PCD mechanism and contribute to the pathogenesis of neurodegenerative diseases such as PD and AD [96]. Recently, researchers have directed their attention to the action of neurotrophic factors in promoting neuronal survival and preventing neurodegenerative diseases. Studies in in vitro models suggest that pretreatment with neurotrophic factors induces the activation of antiapoptotic pathways [33], while tests in in vivo models conclude that administration of neurotrophic factors limits their neuroprotective action due to their poor stability and minimal BBB permeation. Although approaches, such as intraventricular infusion of nerve growth factor in rats after traumatic brain injury, or using IGF1 in clinical trials in patients with amyotrophic lateral sclerosis, resulted in a reduction of neuronal loss, there is not yet an effective treatment for neurodiseases [9].

Working on the hypothesis that the limited survival effects of neurotrophic factors resulting from direct delivery to the brain could be improved by modulating their downstream signaling pathways, various molecular biological approaches have been developed. The advent of microarray technology has led to the development of a genomic approach that has dramatically accelerated efforts to understand the signaling pathways underlying the pro- and antiapoptotic role of neurotrophic factor withdrawal in several in vitro models [19,97]. Microarray technology is supported by a number of bioinformatic and statistical tools applied to genomic data. These tools allow to translate genomic data into information that can assist in understanding the delicate balance between neuronal apoptosis and survival and in identifying genes that showing an altered expression in the promotion and/or protection from apoptosis may represent potential pharmacological targets for already existing drugs. This new therapeutic perspective paves the way toward an innovative pharmacology based on drug targets downstream rather than upstream of membrane receptors, allowing the use of substances acting on key pathways activated by neurotrophic factors.

Despite the considerable progress that has been made in this area, there are still some limitations inherent to microarray technology, as described in the dedicated Section 3. In particular, since there are considerable issues regarding sensitivity and reproducibility of microarray studies, which inevitably cause an high number of false positives, confirmatory techniques, such as proteomic methods, are necessary to test and validate data inferred through the genomic analysis. Proteomics can help to decipher the protein products of genes of interest and to get a clearer picture of the complex regulatory networks that control fundamental biological processes.

The simultaneous monitoring of both RNA and protein expression levels, therefore, may strongly contribute to increasing reliability in drug target discovery and improving the pharmacological approach for counteracting neurodegeneration. In the future, comparing further public transcriptomic and proteomic data inferred across more complex biological models and phenomena will help to deduce useful information about the spectrum of potential drug targets that may give more effective answers for the therapy of neurodegenerative diseases.

Article highlights box

• Neuronal apoptosis is a normal physiological process that is an essential and integral part of neurogenesis. However, its inappropriate activation may contribute to neurodegeneration, a progressive and multifaceted process that results in various chronic disorders, such as Alzheimer’s and Parkinson’s diseases.

• Growth factors are known to be potent agents that counteract neuronal cell death in in vitro models. However, the peptidergic structure of these molecules limits their ability to cross the blood brain barrier (BBB) and exert effective neuroprotection.

• In recent years, the advent of high-throughput technologies has offered a new approach to decipher the transcriptional mechanisms underlying the molecular framework (genes and pathways) of growth factors, unraveling a systems biology based perspective. In particular, in vitro models have been extensively used to study the neuronal apoptotic and/or survival signaling events regulated by growth factors.

• By combining whole genome expression analyses of apoptotic neurons rescued by two very effective growth factors, Igf1 and Pacap, a variety of common genes and functional pathways were found to be statistically deregulated. Several of these deregulated genes encode known drug targets for pharmacological compounds.

• Pharmacological modulation of drug targets in neuroprotective-signaling pathways could be a key to antagonize neurodegeneration. Such a strategy, may focus on the development of pharmacological compounds no longer oriented to emulate the action of growth factors at the membrane receptor level but rather, to modulate their downstream signals.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Acknowledgements

The authors gratefully acknowledge Cristina Calì, Alfia Corsino, Maria Patrizia D’Angelo and Francesco Marino for their administrative and technical assistance.

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Additional information

Funding

The authors are supported by the Italian Ministry of Education, Universities and Research through grant CTN01_00177_817708.