The functional organization of axonal mRNA transport and translation.

Axons extend for tremendously long distances from the neuronal soma and make use of localized mRNA translation to rapidly respond to different extracellular stimuli and physiological states. The locally synthesized proteins support many different functions in both developing and mature axons, raising questions about the mechanisms by which local translation is organized to ensure the appropriate responses to specific stimuli. Publications over the past few years have uncovered new mechanisms for regulating the axonal transport and localized translation of mRNAs, with several of these pathways converging on the regulation of cohorts of functionally related mRNAs - known as RNA regulons - that drive axon growth, axon guidance, injury responses, axon survival and even axonal mitochondrial function. Recent advances point to these different regulatory pathways as organizing platforms that allow the axon's proteome to be modulated to meet its physiological needs.

The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs.

Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3'UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.

Axon Initial Segments Are Required for Efficient Motor Neuron Axon Regeneration and Functional Recovery of Synapses.

The axon initial segment (AIS) generates action potentials and maintains neuronal polarity by regulating the differential trafficking and distribution of proteins, transport vesicles, and organelles. Injury and disease can disrupt the AIS, and the subsequent loss of clustered ion channels and polarity mechanisms may alter neuronal excitability and function. However, the impact of AIS disruption on axon regeneration after injury is unknown. We generated male and female mice with AIS-deficient multipolar motor neurons by deleting AnkyrinG, the master scaffolding protein required for AIS assembly and maintenance. We found that after nerve crush, neuromuscular junction reinnervation was significantly delayed in AIS-deficient motor neurons compared with control mice. In contrast, loss of AnkyrinG from pseudo-unipolar sensory neurons did not impair axon regeneration into the intraepidermal nerve fiber layer. Even after AIS-deficient motor neurons reinnervated the neuromuscular junction, they failed to functionally recover because of reduced synaptic vesicle protein 2 at presynaptic terminals. In addition, mRNA trafficking was disrupted in AIS-deficient axons. Our results show that, after nerve injury, an intact AIS is essential for efficient regeneration and functional recovery of axons in multipolar motor neurons. Our results also suggest that loss of polarity in AIS-deficient motor neurons impairs the delivery of axonal proteins, mRNAs, and other cargoes necessary for regeneration. Thus, therapeutic strategies for axon regeneration must consider preservation or reassembly of the AIS.SIGNIFICANCE STATEMENT Disruption of the axon initial segment is a common event after nervous system injury. For multipolar motor neurons, we show that axon initial segments are essential for axon regeneration and functional recovery after injury. Our results may help explain injuries where axon regeneration fails, and suggest strategies to promote more efficient axon regeneration.

Selective axonal translation of the mRNA isoform encoding prenylated Cdc42 supports axon growth.

The small Rho-family GTPase Cdc42 has long been known to have a role in cell motility and axon growth. The eukaryotic Ccd42 gene is alternatively spliced to generate mRNAs with two different 3' untranslated regions (UTRs) that encode proteins with distinct C-termini. The C-termini of these Cdc42 proteins include CaaX and CCaX motifs for post-translational prenylation and palmitoylation, respectively. Palmitoyl-Cdc42 protein was previously shown to contribute to dendrite maturation, while the prenyl-Cdc42 protein contributes to axon specification and its mRNA was detected in neurites. Here, we show that the mRNA encoding prenyl-Cdc42 isoform preferentially localizes into PNS axons and this localization selectively increases in vivo during peripheral nervous system (PNS) axon regeneration. Functional studies indicate that prenyl-Cdc42 increases axon length in a manner that requires axonal targeting of its mRNA, which, in turn, needs an intact C-terminal CaaX motif that can drive prenylation of the encoded protein. In contrast, palmitoyl-Cdc42 has no effect on axon growth but selectively increases dendrite length. Together, these data show that alternative splicing of the Cdc42 gene product generates an axon growth promoting, locally synthesized prenyl-Cdc42 protein. This article has an associated First Person interview with one of the co-first authors of the paper.

The RNA-Binding Protein HuD Regulates Alternative Splicing and Alternative Polyadenylation in the Mouse Neocortex.

The neuronal Hu/ELAV-like proteins HuB, HuC and HuD are a class of RNA-binding proteins that are crucial for proper development and maintenance of the nervous system. These proteins bind to AU-rich elements (AREs) in the untranslated regions (3'-UTRs) of target mRNAs regulating mRNA stability, transport and translation. In addition to these cytoplasmic functions, Hu proteins have been implicated in alternative splicing and alternative polyadenylation in the nucleus. The purpose of this study was to identify transcriptome-wide effects of HuD deletion on both of these nuclear events using RNA sequencing data obtained from the neocortex of Elavl4-/- (HuD KO) mice. HuD KO affected alternative splicing of 310 genes, including 17 validated HuD targets such as Cbx3, Cspp1, Snap25 and Gria2. In addition, deletion of HuD affected polyadenylation of 53 genes, with the majority of significantly altered mRNAs shifting towards usage of proximal polyadenylation signals (PAS), resulting in shorter 3'-UTRs. None of these genes overlapped with those showing alternative splicing events. Overall, HuD KO had a greater effect on alternative splicing than polyadenylation, with many of the affected genes implicated in several neuronal functions and neuropsychiatric disorders.

Axonal mRNA transport and translation at a glance.

Localization and translation of mRNAs within different subcellular domains provides an important mechanism to spatially and temporally introduce new proteins in polarized cells. Neurons make use of this localized protein synthesis during initial growth, regeneration and functional maintenance of their axons. Although the first evidence for protein synthesis in axons dates back to 1960s, improved methodologies, including the ability to isolate axons to purity, highly sensitive RNA detection methods and imaging approaches, have shed new light on the complexity of the transcriptome of the axon and how it is regulated. Moreover, these efforts are now uncovering new roles for locally synthesized proteins in neurological diseases and injury responses. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of how axonal mRNA transport and translation are regulated, and discuss their emerging links to neurological disorders and neural repair.

hnRNPs Interacting with mRNA Localization Motifs Define Axonal RNA Regulons.

mRNA translation in axons enables neurons to introduce new proteins at sites distant from their cell body. mRNA-protein interactions drive this post-transcriptional regulation, yet knowledge of RNA binding proteins (RBP) in axons is limited. Here we used proteomics to identify RBPs interacting with the axonal localizing motifs of Nrn1, Hmgb1, Actb, and Gap43 mRNAs, revealing many novel RBPs in axons. Interestingly, no RBP is shared between all four RNA motifs, suggesting graded and overlapping specificities of RBP-mRNA pairings. A systematic assessment of axonal mRNAs interacting with hnRNP H1, hnRNP F, and hnRNP K, proteins that bound with high specificity to Nrn1 and Hmgb1, revealed that axonal mRNAs segregate into axon growth-associated RNA regulons based on hnRNP interactions. Axotomy increases axonal transport of hnRNPs H1, F, and K, depletion of these hnRNPs decreases axon growth and reduces axonal mRNA levels and axonal protein synthesis. Thus, subcellular hnRNP-interacting RNA regulons support neuronal growth and regeneration.

Axonal localization of neuritin/CPG15 mRNA is limited by competition for HuD binding.

HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.

mRNAs and Protein Synthetic Machinery Localize into Regenerating Spinal Cord Axons When They Are Provided a Substrate That Supports Growth.

Although intra-axonal protein synthesis is well recognized in cultured neurons and during development in vivo, there have been few reports of mRNA localization and/or intra-axonal translation in mature CNS axons. Indeed, previous work indicated that mature CNS axons contain much lower quantities of translational machinery than PNS axons, leading to the conclusion that the capacity for intra-axonal protein synthesis is linked to the intrinsic capacity of a neuron for regeneration, with mature CNS neurons showing much less growth after injury than PNS neurons. However, when regeneration by CNS axons is facilitated, it is not known whether the intra-axonal content of translational machinery changes or whether mRNAs localize into these axons. Here, we have used a peripheral nerve segment grafted into the transected spinal cord of adult rats as a supportive environment for regeneration by ascending spinal axons. By quantitative fluorescent in situ hybridization combined with immunofluorescence to unambiguously distinguish intra-axonal mRNAs, we show that regenerating spinal cord axons contain ß-actin, GAP-43, Neuritin, Reg3a, Hamp, and Importin ß1 mRNAs. These axons also contain 5S rRNA, phosphorylated S6 ribosomal protein, eIF2α translation factor, and 4EBP1 translation factor inhibitory protein. Different levels of these mRNAs in CNS axons from regenerating PNS axons may relate to differences in the growth capacity of these neurons, although the presence of mRNA transport and likely local translation in both CNS and PNS neurons suggests an active role in the regenerative process. SIGNIFICANCE STATEMENT: Although peripheral nerve axons retain the capacity to locally synthesize proteins into adulthood, previous studies have argued that mature brain and spinal cord axons cannot synthesize proteins. Protein synthesis in peripheral nerve axons is increased during regeneration, and intra-axonally synthesized proteins have been shown to contribute to nerve regeneration. Here, we show that mRNAs and translational machinery are transported into axons regenerating from the spinal cord into the permissive environment of a peripheral nerve graft. Our data raise the possibility that spinal cord axons may make use of localized protein synthesis for regeneration.

Axonal amphoterin mRNA is regulated by translational control and enhances axon outgrowth.

High mobility group (HMG) proteins concentrate in the nucleus, interacting with chromatin. Amphoterin is an HMG protein (HMGB1) that has been shown to have extranuclear functions and can be secreted from some cell types. Exogenous amphoterin can increase neurite growth, suggesting that the secreted protein may have growth promoting activities in neurons. Consistent with this, we show that depletion of amphoterin mRNA from cultured adult rat DRG neurons attenuates neurite outgrowth, pointing to autocrine or paracrine mechanisms for its growth-promoting effects. The mRNA encoding amphoterin localizes to axonal processes and we showed recently that its 3'-UTR is sufficient for axonal localization of heterologous transcripts (Donnelly et al., 2013). Here, we show that amphoterin mRNA is transported constitutively into axons of adult DRG neurons. A preconditioning nerve injury increases the levels of amphoterin protein in axons without a corresponding increase in amphoterin mRNA in the axons. A 60 nucleotide region of the amphoterin mRNA 3'-UTR is necessary and sufficient for its localization into axons of cultured sensory neurons. Amphoterin mRNA 3'-UTR is also sufficient for axonal localization in distal axons of DRG neurons in vivo. Overexpression of axonally targeted amphoterin mRNA increases axon outgrowth in cultured sensory neurons, but axon growth is not affected when the overexpressed mRNA is restricted to the cell body.

Axonal transcription factors signal retrogradely in lesioned peripheral nerve.

Retrograde axonal injury signalling stimulates cell body responses in lesioned peripheral neurons. The involvement of importins in retrograde transport suggests that transcription factors (TFs) might be directly involved in axonal injury signalling. Here, we show that multiple TFs are found in axons and associate with dynein in axoplasm from injured nerve. Biochemical and functional validation for one TF family establishes that axonal STAT3 is locally translated and activated upon injury, and is transported retrogradely with dynein and importin α5 to modulate survival of peripheral sensory neurons after injury. Hence, retrograde transport of TFs from axonal lesion sites provides a direct link between axon and nucleus.

Dynamic Changes in Local Protein Synthetic Machinery in Regenerating Central Nervous System Axons after Spinal Cord Injury.

Intra-axonal localization of mRNAs and protein synthesis machinery (PSM) endows neurons with the capacity to generate proteins locally, allowing precise spatiotemporal regulation of the axonal response to extracellular stimuli. A number of studies suggest that this local translation is a promising target to enhance the regenerative capacity of damaged axons. Using a model of central nervous system (CNS) axons regenerating into intraspinal peripheral nerve grafts (PNGs) we established that adult regenerating CNS axons contain several different mRNAs and protein synthetic machinery (PSM) components in vivo. After lower thoracic level spinal cord transection, ascending sensory axons regenerate into intraspinal PNGs but axon growth is stalled when they reach the distal end of the PNG (3 versus 7 weeks after grafting, resp.). By immunofluorescence with optical sectioning of axons by confocal microscopy, the total and phosphorylated forms of PSMs are significantly lower in stalled compared with actively regenerating axons. Reinjury of these stalled axons increased axonal localization of the PSM proteins, indicative of possible priming for a subcellular response to axotomy. These results suggest that axons downregulate protein synthetic capacity as they cease growing, yet they retain the ability to upregulate PSM after a second injury.

Calcium release from intra-axonal endoplasmic reticulum leads to axon degeneration through mitochondrial dysfunction.

Axonal degeneration represents an early pathological event in neurodegeneration, constituting an important target for neuroprotection. Regardless of the initial injury, which could be toxic, mechanical, metabolic, or genetic, degeneration of axons shares a common mechanism involving mitochondrial dysfunction and production of reactive oxygen species. Critical steps in this degenerative process are still unknown. Here we show that calcium release from the axonal endoplasmic reticulum (ER) through ryanodine and IP3 channels activates the mitochondrial permeability transition pore and contributes to axonal degeneration triggered by both mechanical and toxic insults in ex vivo and in vitro mouse and rat model systems. These data reveal a critical and early ER-dependent step during axonal degeneration, providing novel targets for axonal protection in neurodegenerative conditions.

Limited availability of ZBP1 restricts axonal mRNA localization and nerve regeneration capacity.

Subcellular localization of mRNAs is regulated by RNA-protein interactions. Here, we show that introduction of a reporter mRNA with the 3'UTR of ß-actin mRNA competes with endogenous mRNAs for binding to ZBP1 in adult sensory neurons. ZBP1 is needed for axonal localization of ß-actin mRNA, and introducing GFP with the 3'UTR of ß-actin mRNA depletes axons of endogenous ß-actin and GAP-43 mRNAs and attenuates both in vitro and in vivo regrowth of severed axons. Consistent with limited levels of ZBP1 protein in adult neurons, mice heterozygous for the ZBP1 gene are haploinsufficient for axonal transport of ß-actin and GAP-43 mRNAs and for regeneration of peripheral nerve. Exogenous ZBP1 can rescue the RNA transport deficits, but the axonal growth deficit is only rescued if the transported mRNAs are locally translated. These data support a direct role for ZBP1 in transport and translation of mRNA cargos in axonal regeneration in vitro and in vivo.

Axonal transport of neural membrane protein 35 mRNA increases axon growth.

Many neuronal mRNAs are transported from cell bodies into axons and dendrites. Localized translation of the mRNAs brings autonomy to these processes that can be vast distances from the cell body. For axons, these translational responses have been linked to growth and injury signaling, but there has been little information about local function of individual axonally synthesized proteins. In the present study, we show that axonal injury increases levels of the mRNA encoding neural membrane protein 35 (NMP35) in axons, with a commensurate decrease in the cell body levels of NMP35 mRNA. The 3' untranslated region (3'UTR) of NMP35 is responsible for this localization into axons. Previous studies have shown that NMP35 protein supports cell survival by inhibiting Fas-ligand-mediated apoptosis; however, these investigations did not distinguish functions of the locally generated NMP35 protein. Using axonally targeted versus cell-body-restricted NMP35 constructs, we show that NMP35 supports axonal growth, and overexpression of an axonally targeted NMP35 mRNA is sufficient to increase axonal outgrowth.

Axonal localization of neuritin/CPG15 mRNA in neuronal populations through distinct 5' and 3' UTR elements.

Many neuronal mRNAs are actively transported into distal axons. The 3' untranslated regions (UTRs) of axonal mRNAs often contain cues for their localization. The 3' UTR of neuritin mRNA was shown to be sufficient for localization into axons of hippocampal neurons. Here, we show that neuritin mRNA localizes into axons of rat sensory neurons, but this is predominantly driven by the 5' rather than 3' UTR. Neuritin mRNA shifts from cell body to axon predominantly after nerve crush injury, suggesting that it encodes a growth-associated protein. Consistent with this, overexpression of neuritin increases axon growth but only when its mRNA localizes into the axons.

Axonally synthesized ß-actin and GAP-43 proteins support distinct modes of axonal growth.

Increasing evidence points to the importance of local protein synthesis for axonal growth and responses to axotomy, yet there is little insight into the functions of individual locally synthesized proteins. We recently showed that expression of a reporter mRNA with the axonally localizing ß-actin mRNA 3'UTR competes with endogenous ß-actin and GAP-43 mRNAs for binding to ZBP1 and axonal localization in adult sensory neurons (Donnelly et al., 2011). Here, we show that the 3'UTR of GAP-43 mRNA can deplete axons of endogenous ß-actin mRNA. We took advantage of this 3'UTR competition to address the functions of axonally synthesized ß-actin and GAP-43 proteins. In cultured rat neurons, increasing axonal synthesis of ß-actin protein while decreasing axonal synthesis of GAP-43 protein resulted in short highly branched axons. Decreasing axonal synthesis of ß-actin protein while increasing axonal synthesis of GAP-43 protein resulted in long axons with few branches. siRNA-mediated depletion of overall GAP-43 mRNA from dorsal root ganglia (DRGs) decreased the length of axons, while overall depletion of ß-actin mRNA from DRGs decreased the number of axon branches. These deficits in axon growth could be rescued by transfecting with siRNA-resistant constructs encoding ß-actin or GAP-43 proteins, but only if the mRNAs were targeted for axonal transport. Finally, in ovo electroporation of axonally targeted GAP-43 mRNA increased length and axonally targeted ß-actin mRNA increased branching of sensory axons growing into the chick spinal cord. These studies indicate that axonal translation of ß-actin mRNA supports axon branching and axonal translation of GAP-43 mRNA supports elongating growth.

Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits.

Spinal muscular atrophy (SMA), caused by the deletion of the SMN1 gene, is the leading genetic cause of infant mortality. SMN protein is present at high levels in both axons and growth cones, and loss of its function disrupts axonal extension and pathfinding. SMN is known to associate with the RNA-binding protein hnRNP-R, and together they are responsible for the transport and/or local translation of ß-actin mRNA in the growth cones of motor neurons. However, the full complement of SMN-interacting proteins in neurons remains unknown. Here we used mass spectrometry to identify HuD as a novel neuronal SMN-interacting partner. HuD is a neuron-specific RNA-binding protein that interacts with mRNAs, including candidate plasticity-related gene 15 (cpg15). We show that SMN and HuD form a complex in spinal motor axons, and that both interact with cpg15 mRNA in neurons. CPG15 is highly expressed in the developing ventral spinal cord and can promote motor axon branching and neuromuscular synapse formation, suggesting a crucial role in the development of motor axons and neuromuscular junctions. Cpg15 mRNA previously has been shown to localize into axonal processes. Here we show that SMN deficiency reduces cpg15 mRNA levels in neurons, and, more importantly, cpg15 overexpression partially rescues the SMN-deficiency phenotype in zebrafish. Our results provide insight into the function of SMN protein in axons and also identify potential targets for the study of mechanisms that lead to the SMA pathology and related neuromuscular diseases.

Enabling Systemic Identification and Functionality Profiling for Cdc42 Homeostatic Modulators.

Homeostatic modulation is pivotal in modern therapeutics. However, the discovery of bioactive materials to achieve this functionality is often random and unpredictive. Here, we enabled a systemic identification and functional classification of chemicals that elicit homeostatic modulation of signaling through Cdc42, a classical small GTPase of Ras superfamily. Rationally designed for high throughput screening, the capture of homeostatic modulators (HMs) along with molecular re-docking uncovered at least five functionally distinct classes of small molecules. This enabling led to partial agonists, hormetic agonists, bona fide activators and inhibitors, and ligand-enhanced agonists. Novel HMs exerted striking functionality in bradykinin-Cdc42 activation of actin remodelingand modified Alzheimer's disease-like behavior in mouse model. This concurrent computer-aided and experimentally empowered HM profiling highlights a model path for predicting HM landscape. One Sentence Summary: With concurrent experimental biochemical profiling and in silico computer-aided drug discovery (CADD) analysis, this study enabled a systemic identification and holistic classification of Cdc42 homeostatic modulators (HMs) and demonstrated the power of CADD to predict HM classes that can mimic the pharmacological functionality of interests. Introduction: Maintainingbody homeostasisis the ultimate keyto health. Thereare rich resources of bioactive materials for this functionality from both natural and synthetic chemical repertories including partial agonists (PAs) and various allosteric modulators. These homeostatic modulators (HMs) play a unique role in modern therapeutics for human diseases such as mental disorders and drug addiction. Buspirone, for example, acts as a PA for serotonin 5-HT 1A receptor but is an antagonist of the dopamine D 2 receptor. Such medical useto treat general anxietydisorders (GADs) has become one of the most-commonly prescribed medications. However, most HMs in current uses target membrane proteins and are often derived from random discoveries. HMs as therapeutics targeting cytoplasmic proteins are even more rare despite that they are in paramount needs (e. g. targeting Ras superfamily small GTPases). Rationale: Cdc42, a classical member of small GTPases of Ras superfamily, regulates PI3K-AKT and Raf-MEK-ERK pathways and has been implicated in various neuropsychiatric and mental disorders as well as addictive diseases and cancer. We previously reported the high-throughput in-silico screening followed by biological characterization of novel small molecule modulators (SMMs) of Cdc42-intersectin (ITSN) protein-protein interactions (PPIs). Based on a serendipitously discovered SMM ZCL278 with PA profile as a model compound, we hypothesized that there are more varieties of such HMs of Cdc42 signaling, and the model HMs can be defined by their distinct Cdc42-ITSN binding mechanisms using computer-aided drug discovery (CADD) analysis. We further reasoned that molecular modeling coupled with experimental profiling can predict HM spectrum and thus open the door for the holistic identification and classification of multifunctional cytoplasmic target-dependent HMs as therapeutics. Results: The originally discovered Cdc42 inhibitor ZCL278 displaying PA properties prompted the inquiry whether this finding represented a random encounter of PAs or whether biologically significant PAs can be widely present. The top ranked compounds were initially defined by structural fitness and binding scores to Cdc42. Because higher binding scores do not necessarily translate to higher functionality, we performed exhaustive experimentations with over 2,500 independent Cdc42-GEF (guanine nucleotide exchange factor) assays to profile the GTP loading activities on all 44 top ranked compounds derived from the SMM library. The N-MAR-GTP fluorophore-based Cdc42-GEF assay platform provided the first glimpse of the breadth of HMs. A spectrum of Cdc42 HMs was uncovered that can be categorized into five functionally distinct classes: Class I-partial competitive agonists, Class II-hormetic agonists, Class III- bona fide inhibitors (or inverse agonists), Class IV- bona fide activators or agonists, and Class V-ligand-enhanced agonists. Remarkably, model HMs such as ZCL278, ZCL279, and ZCL367 elicited striking biological functionality in bradykinin-Cdc42 activation of actin remodeling and modified Alzheimer's disease (AD)-like behavior in mouse model. Concurrently, we applied Schrödinger-enabled analyses to perform CADD predicted classification of Cdc42 HMs. We modified the classic molecular docking to instill a preferential binding pocket order (PBPO) of Cdc42-ITSN, which was based on the five binding pockets in interface of Cdc42-ITSN. We additionally applied a structure-based pharmacophore hypothesis generation for the model compounds. Then, using Schrödinger's Phase Shape, 3D ligand alignments assigned HMs to Class I, II, III, IV, and V compounds. In this HM library compounds, PBPO, matching pharmacophoric featuring, and shape alignment, all put ZCL993 in Class II compound category, which was confirmed in the Cdc42-GEF assay. Conclusion: HMs can target diseased cells or tissues while minimizing impacts on tissues that are unaffected. Using Cdc42 HM model compounds as a steppingstone, GTPase activation-based screening of SMM library uncovered five functionally distinct Cdc42 HM classes among which novel efficacies towards alleviating dysregulated AD-like features in mice were identified. Furthermore, molecular re-docking of HM model compounds led to the concept of PBPO. The CADD analysis with PBPO revealed similar profile in a color-coded spectrum to these five distinct classes of Cdc42 HMs identified by biochemical functionality-based screening. The current study enabled a systemic identification and holistic classification of Cdc42 HMs and demonstrated the power of CADD to predict an HM category that can mimic the pharmacological functionality of interests. With artificial intelligence/machine learning (AI/ML) on the horizon to mirror experimental pharmacological discovery like AlphaFold for protein structure prediction, our study highlights a model path to actively capture and profile HMs in potentially any PPI landscape. Identification and functional classification of Cdc42 homeostatic modulators HMs: Using Cdc42 HM model compounds as reference, GTPase activation-based screening of compound libraries uncovered five functionally distinct Cdc42 HM classes. HMs showed novel efficacies towards alleviating dysregulated Alzheimer's disease (AD)-like behavioral and molecular deficits. In parallel, molecular re-docking of HM model compounds established their preferential binding pocket orders (PBPO). PBPO-based profiling (Red reflects the most, whereas green reflects the least, preferable binding pocket) revealed trends of similar pattern to the five classes from the functionality-based classification.