Interactions between Ras and Rap signaling pathways during neurodevelopment in health and disease.

The Ras family of small GTPases coordinates tissue development by modulating cell proliferation, cell-cell adhesion, and cellular morphology. Perturbations of any of these key steps alter nervous system development and are associated with neurological disorders. While the underlying causes are not known, genetic mutations in Ras and Rap GTPase signaling pathways have been identified in numerous neurodevelopmental disorders, including autism spectrum, neurofibromatosis, intellectual disability, epilepsy, and schizophrenia. Despite diverse clinical presentations, intersections between these two signaling pathways may provide a better understanding of how deviations in neurodevelopment give rise to neurological disorders. In this review, we focus on presynaptic and postsynaptic functions of Ras and Rap GTPases. We highlight various roles of these small GTPases during synapse formation and plasticity. Based on genomic analyses, we discuss how disease-related mutations in Ras and Rap signaling proteins may underlie human disorders. Finally, we discuss how recent observations have identified molecular interactions between these pathways and how these findings may provide insights into the mechanisms that underlie neurodevelopmental disorders.

PXF-1 promotes synapse development at the neuromuscular junction in Caenorhabditis elegans.

Guanine nucleotide exchange factors (GEFs) are a family of proteins that modulate small G protein signaling. Mutations in a subfamily of GEFs that act on Rap, known as RapGEFs, have been associated with neurological disorders, and knockout mice display impairments in neuronal activity. However, the precise functions of RapGEFs in the nervous system remain unclear. Here, we have used the Caenorhabditis elegans neuromuscular junction, to investigate how the RapGEF homolog, PXF-1, regulates synaptic function. We found that loss of function mutations in pxf-1 reduced cholinergic activity at the neuromuscular junction. We observed that PXF-1 is expressed in the nervous system, and its expression in neurons is sufficient to promote synaptic activity. In pxf-1 mutant animals, there is a reduction in the levels of synaptic vesicles in cholinergic motor neurons but no change in the overall synapse numbers. In addition to synaptic vesicles proteins, we also found that filamentous actin, a scaffold for nascent synapses, was reduced at developing cholinergic synapses in pxf-1 mutant animals. Our studies indicate that PXF-1 regulates neuromuscular function by promoting the formation of actin filaments to support the development of motor neuron synapses.

C. elegans MAGU-2/Mpp5 homolog regulates epidermal phagocytosis and synapse density.

Synapses are dynamic connections that underlie essential functions of the nervous system. The addition, removal, and maintenance of synapses govern the flow of information in neural circuits throughout the lifetime of an animal. While extensive studies have elucidated many intrinsic mechanisms that neurons employ to modulate their connections, increasing evidence supports the roles of non-neuronal cells, such as glia, in synapse maintenance and circuit function. We previously showed that C. elegans epidermis regulates synapses through ZIG-10, a cell-adhesion protein of the immunoglobulin domain superfamily. Here we identified a member of the Pals1/MPP5 family, MAGU-2, that functions in the epidermis to modulate phagocytosis and the number of synapses by regulating ZIG-10 localization. Furthermore, we used light and electron microscopy to show that this epidermal mechanism removes neuronal membranes from the neuromuscular junction, dependent on the conserved phagocytic receptor CED-1. Together, our study shows that C. elegans epidermis constrains synaptic connectivity, in a manner similar to astrocytes and microglia in mammals, allowing optimized output of neural circuits.

Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.

Novel Mutations in Synaptic Transmission Genes Suppress Neuronal Hyperexcitation in Caenorhabditis elegans.

Acetylcholine (ACh) receptors (AChR) regulate neural circuit activity in multiple contexts. In humans, mutations in ionotropic acetylcholine receptor (iAChR) genes can cause neurological disorders, including myasthenia gravis and epilepsy. In Caenorhabditis elegans, iAChRs play multiple roles in the locomotor circuit. The cholinergic motor neurons express an ACR-2-containing pentameric AChR (ACR-2R) comprised of ACR-2, ACR-3, ACR-12, UNC-38, and UNC-63 subunits. A gain-of-function mutation in the non-α subunit gene acr-2 [acr-2(gf)] causes defective locomotion as well as spontaneous convulsions. Previous studies of genetic suppressors of acr-2(gf) have provided insights into ACR-2R composition and assembly. Here, to further understand how the ACR-2R regulates neuronal activity, we expanded the suppressor screen for acr-2(gf)-induced convulsions. The majority of these suppressor mutations affect genes that play critical roles in synaptic transmission, including two novel mutations in the vesicular ACh transporter unc-17 In addition, we identified a role for a conserved major facilitator superfamily domain (MFSD) protein, mfsd-6, in regulating neural circuit activity. We further defined a role for the sphingosine (SPH) kinase (Sphk) sphk-1 in cholinergic neuron activity, independent of previously known signaling pathways. Overall, the genes identified in our study suggest that optimal modulation of synaptic activity is balanced by the differential activities of multiple pathways, and the novel alleles provide valuable reagents to further dissect neuronal mechanisms regulating the locomotor circuit.

Asynchronous Cholinergic Drive Correlates with Excitation-Inhibition Imbalance via a Neuronal Ca2+ Sensor Protein.

Excitation-inhibition imbalance in neural networks is widely linked to neurological and neuropsychiatric disorders. However, how genetic factors alter neuronal activity, leading to excitation-inhibition imbalance, remains unclear. Here, using the C. elegans locomotor circuit, we examine how altering neuronal activity for varying time periods affects synaptic release pattern and animal behavior. We show that while short-duration activation of excitatory cholinergic neurons elicits a reversible enhancement of presynaptic strength, persistent activation results to asynchronous and reduced cholinergic drive, inducing imbalance between endogenous excitation and inhibition. We find that the neuronal calcium sensor protein NCS-2 is required for asynchronous cholinergic release in an activity-dependent manner and dampens excitability of inhibitory neurons non-cell autonomously. The function of NCS-2 requires its Ca2+ binding and membrane association domains. These results reveal a synaptic mechanism implicating asynchronous release in regulation of excitation-inhibition balance.

A Two-Immunoglobulin-Domain Transmembrane Protein Mediates an Epidermal-Neuronal Interaction to Maintain Synapse Density.

Synaptic maintenance is essential for neural circuit function. In the C. elegans locomotor circuit, motor neurons are in direct contact with the epidermis. Here, we reveal a novel epidermal-neuronal interaction mediated by a two-immunoglobulin domain transmembrane protein, ZIG-10, that is necessary for maintaining cholinergic synapse density. ZIG-10 is localized at the cell surface of epidermis and cholinergic motor neurons, with high levels at areas adjacent to synapses. Loss of zig-10 increases the number of cholinergic excitatory synapses and exacerbates convulsion behavior in a seizure model. Mis-expression of zig-10 in GABAergic inhibitory neurons reduces GABAergic synapse number, dependent on the presence of ZIG-10 in the epidermis. Furthermore, ZIG-10 interacts with the tyrosine kinase SRC-2 to regulate the phagocytic activity of the epidermis to restrict cholinergic synapse number. Our studies demonstrate the highly specific roles of non-neuronal cells in modulating neural circuit function, through neuron-type-specific maintenance of synapse density.

Advances in synapse formation: forging connections in the worm.

UNLABELLED: Synapse formation is the quintessential process by which neurons form specific connections with their targets to enable the development of functional circuits. Over the past few decades, intense research efforts have identified thousands of proteins that localize to the pre- and postsynaptic compartments. Genetic dissection has provided important insights into the nexus of the molecular and cellular network, and has greatly advanced our knowledge about how synapses form and function physiologically. Moreover, recent studies have highlighted the complex regulation of synapse formation with the identification of novel mechanisms involving cell interactions from non-neuronal sources. In this review, we cover the conserved pathways required for synaptogenesis and place specific focus on new themes of synapse modulation arising from studies in Caenorhabditis elegans. For further resources related to this article, please visit the WIREs website. CONFLICT OF INTEREST: The authors have declared no conflicts of interest for this article.

ERK-mediated phosphorylation of TFAM downregulates mitochondrial transcription: implications for Parkinson's disease.

Mitochondrial transcription factor A (TFAM) regulates mitochondrial biogenesis, which is downregulated by extracellular signal-regulated protein kinases (ERK1/2) in cells treated chronically with the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+). We utilized mass spectrometry to identify ERK1/2-dependent TFAM phosphorylation sites. Mutation of TFAM at serine 177 to mimic phosphorylation recapitulated the effects of MPP+ in decreasing the binding of TFAM to the light strand promoter, suppressing mitochondrial transcription. Mutant TFAM was unable to affect respiratory function or rescue the effects of MPP+ on respiratory complexes. These data disclose a novel mechanism by which ERK1/2 regulates mitochondrial function through direct phosphorylation of TFAM.

Mutant LRRK2 elicits calcium imbalance and depletion of dendritic mitochondria in neurons.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with familial and sporadic cases of Parkinson disease. Mutant LRRK2 causes in vitro and in vivo neurite shortening, mediated in part by autophagy, and a parkinsonian phenotype in transgenic mice; however, the underlying mechanisms remain unclear. Because mitochondrial content/function is essential for dendritic morphogenesis and maintenance, we investigated whether mutant LRRK2 affects mitochondrial homeostasis in neurons. Mouse cortical neurons expressing either LRRK2 G2019S or R1441C mutations exhibited autophagic degradation of mitochondria and dendrite shortening. In addition, mutant LRRK2 altered the ability of the neurons to buffer intracellular calcium levels. Either calcium chelators or inhibitors of voltage-gated L-type calcium channels prevented mitochondrial degradation and dendrite shortening. These data suggest that mutant LRRK2 causes a deficit in calcium homeostasis, leading to enhanced mitophagy and dendrite shortening.

Regulation of the autophagy protein LC3 by phosphorylation.

Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP(+)) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease-associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl-cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity.

Mitochondrial autophagy as a compensatory response to PINK1 deficiency.

Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.

Loss of PINK1 function promotes mitophagy through effects on oxidative stress and mitochondrial fission.

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.

Autophagy in neurite injury and neurodegeneration: in vitro and in vivo models.

Recent advances indicate that maintaining a balanced level of autophagy is critically important for neuronal health and function. Pathologic dysregulation of macroautophagy has been implicated in synaptic dysfunction, cellular stress, and neuronal cell death. Autophagosomes and autolysosomes are induced in acute and chronic neurological disorders including stroke, brain trauma, neurotoxin injury, Parkinson's, Alzheimer's, Huntington's, motor neuron, prion, lysosomal storage, and other neurodegenerative diseases. Compared to other cell types, neuronal autophagy research presents particular challenges that may be addressed through still evolving techniques. Neuronal function depends upon maintenance of axons and dendrites (collectively known as neurites) that extend for great distances from the cell body. Both autophagy and mitochondrial content have been implicated in regulation of neurite length and function in physiological (plasticity) and pathological remodeling. Here, we highlight several molecular cell biological and imaging methods to study autophagy and mitophagy in neuritic and somatic compartments of differentiated neuronal cell lines and primary neuron cultures, using protocols developed in toxic and genetic models of parkinsonian neurodegeneration. In addition, mature neurons can be studied using in vivo protocols for modeling ischemic and traumatic injuries. Future challenges include application of automated computer-assisted image analysis to the axodendritic tree of individual neurons and improving methods for measuring neuronal autophagic flux.

Autophagy in neuroprotection and neurodegeneration: A question of balance.

A central issue in developing therapies for neurodegenerative diseases involves understanding why adaptive responses to stress or injury fail to prevent synaptic dysfunction and neuronal cell death. Macroautophagy is a major, evolutionarily conserved response to nutrient and bioenergetic stresses, which has the capacity to remove aggregated proteins and damaged organelles such as mitochondria. This has prompted intense interest in autophagy-related therapies for Huntington's, Alzheimer's, Parkinson's, stroke and other neurological diseases. However, excessive or imbalanced induction of autophagic recycling can actively contribute to neuronal atrophy, neurite degeneration and cell death. Oxidative-, aging- and disease-related increase in demand for autophagy, coupled with declining axonal trafficking, lysosomal degradation or biosynthetic efficiencies promote increased susceptibility to a harmful state of autophagic stress. A more complete understanding of dysfunction along the entire spectrum of autophagic recycling, from autophagosome formation through clearance and regeneration of new cellular components is necessary to restore balance to the system, promote neuronal health and maximize therapeutic potentials.

Role of autophagy in G2019S-LRRK2-associated neurite shortening in differentiated SH-SY5Y cells.

Neuritic retraction represents a prominent feature of the degenerative phenotype associated with mutations in leucine rich repeat kinase 2 (LRRK2) that are implicated in autosomal dominant and some cases of sporadic Parkinson's disease. Alterations in macroautophagy, the vacuolar catabolism of cytoplasmic constituents, have been described in Parkinson's disease. In this study, we utilized retinoic-acid differentiated SH-SY5Y cells to determine whether autophagy contributes to mutant LRRK2-associated neurite degeneration. Transfection of pre-differentiated SH-SY5Y cells with LRRK2 cDNA containing the common G2019S mutation resulted in significant decreases in neurite length, which were not observed in cells transfected with wild type LRRK2 or its kinase-dead K1906M mutation. G2019S LRRK2 transfected cells also exhibited striking increases in autophagic vacuoles in both neuritic and somatic compartments, as demonstrated by fluorescence and western blot analysis of the autophagy marker green fluorescent protein-tagged microtubule-associated protein Light Chain 3 and by transmission electron microscopy. RNA interference knockdown of LC3 or Atg7, two essential components of the conserved autophagy machinery, reversed the effects of G2019S LRRK2 expression on neuronal process length, whereas rapamycin potentiated these effects. The mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) inhibitor 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126) reduced LRRK2-induced neuritic autophagy and neurite shortening, implicating MAPK/ERK-related signaling. These results indicate an active role for autophagy in neurite remodeling induced by pathogenic mutation of LRRK2.

Intravascular pressure and diameter profile of the utero-ovarian resistance artery network: estrous cycle-dependent modulation of resistance artery tone.

Blood flow to the ovary varies dramatically in both magnitude and distribution throughout the estrous cycle to meet the hormonal and metabolic demands of the ovarian parenchyma as it cyclically develops and regresses. Several vascular components appear to be critical to vascular regulation of the ovary. As a first step in resolving the role of the resistance arteries and their paired veins in regulating ovarian blood flow and transvascular exchange, we characterized the architecture and intravascular pressure profile of the utero-ovarian resistance artery network in an in vivo preparation of the ovary of the anesthetized Golden hamster. We also investigated estrous cycle-dependent changes in resistance artery tone. The right ovary and the cranial aspect of the uterus in 26 female hamsters were exposed for microcirculatory observations. Estrous-cycle phase was determined in each animal before experimentation. The utero-ovarian vascular architecture was determined and resistance artery diameters were measured in each animal by video microscopy. Servo-null intravascular pressure measurements were made throughout the uteroovarian arterial network in 11 of the animals. Architectural data showed a complex anastomotic network jointly supplying the uterus and ovary. Resistance arteries showed a high degree of coiling and close apposition to veins, maximizing countercurrent-exchange capabilities. Arterial pressure dropped below 60% of systemic arterial pressure before the arteries entered the ovary. Both the ovarian artery and the uterine artery, which jointly feed the ovary, showed cycle day-dependent changes in diameter. Arterial diameters were smallest on the day following ovulation, during the brief luteal phase of the hamster. The data show that resistance arteries comprise a critical part of a complex network designed for intimate local communication and control and suggest that these arteries may play an important role in regulating ovarian blood flow in an estrous cycle-specific manner.