Circulating Cell-Free Mitochondrial DNA and Depressive Symptoms Among Low-Active Adults Who Smoke.

OBJECTIVES: Mitochondrial dysfunction is implicated in the pathophysiology of psychiatric disorders. Levels of circulating cell-free mitochondrial DNA (cf-mtDNA) are observed to be altered in depression. However, the few studies that have measured cf-mtDNA in depression have reported conflicting findings. This study examined cf-mtDNA and depressive symptoms in low-active adults who smoke. METHODS: Participants were adults 18 to 65 years old ( N = 109; 76% female) with low baseline physical activity and depressive symptoms recruited for a smoking cessation study. Self-report measures assessed depression severity, positive and negative affect, and behavioral activation. Blood was collected and analyzed for cf-mtDNA. Relationships between depressive symptoms and cf-mtDNA were examined with correlations and linear regression. RESULTS: Levels of cf-mtDNA were associated with categorically defined depression (Center for Epidemiologic Studies Depression Scale score >15), lower positive affect, and decreased behavioral activation ( p < .05). Relationships remained significant after adjustment for age, sex, and nicotine dependence. In a linear regression model including all depressive symptom measures as predictors, Center for Epidemiologic Studies Depression Scale group and lower positive affect remained significant. CONCLUSIONS: This work suggests that mitochondrial changes are associated with depressive symptoms in low-active adults who smoke. Higher levels of cf-mtDNA in association with depression and with lower positive affect and decreased behavioral activation are consistent with a possible role for mitochondrial function in depressive symptoms.

Examining the Biological Impacts of Parent-Child Relationship Dynamics on Preschool-Aged Children who have Experienced Adversity.

Parent-child relationship dynamics have been shown to predict socioemotional and behavioral outcomes for children, but little is known about how they may affect biological development. The aim of this study was to test if observational assessments of parent-child relationship dynamics (cohesion, enmeshment, and disengagement) were associated with three biological indices of early life adversity and downstream health risk: (1) methylation of the glucocorticoid receptor gene (NR3C1), (2) telomere attrition, and (3) mitochondrial biogenesis, indexed by mitochondrial DNA copy number (mtDNAcn), all of which were measured in children's saliva. We tested hypotheses using a sample of 254 preschool-aged children (M age = 51.04 months) with and without child welfare-substantiated maltreatment (52% with documented case of moderate-severe maltreatment) who were racially and ethnically diverse (17% Black, 40% White, 23% biracial, and 20% other races; 45% Hispanic) and from primarily low-income backgrounds (91% qualified for public assistance). Results of path analyses revealed that: (1) higher parent-child cohesion was associated with lower levels of methylation of NR3C1 exon 1D and longer telomeres, and (2) higher parent-child disengagement was associated with higher levels of methylation of NR3C1 exon 1D and shorter telomeres. Results suggest that parent-child relationship dynamics may have distinct biological effects on children.

Postsynaptic Mechanisms Render Syn I/II/III Mice Highly Responsive to Psychostimulants.

BACKGROUND: Synapsins are encoded by SYN I, SYN II, and SYN III, and they regulate neurotransmitter release by maintaining a reserve pool of synaptic vesicles. METHODS: Presynaptic dopamine responses to cocaine were examined by microdialysis, and postsynaptic responses were evaluated to various dopamine receptor agonists in the open field with SynI/SynII/SynIII triple knockout mice. RESULTS: Triple knockout mice showed enhanced spontaneous locomotion in a novel environment and were hyper-responsive to indirect and direct D1 and D2 dopamine agonists. Triple knockout animals appeared sensitized to cocaine upon first open field exposure; sensitization developed across days in wild-type controls. When mutants were preexposed to a novel environment before injection, cocaine-stimulated locomotion was reduced and behavioral sensitization retarded. Baseline dopamine turnover was enhanced in mutants and novel open field exposure increased their striatal dopamine synthesis rates. As KCl-depolarization stimulated comparable dopamine release in both genotypes, their readily releasable pools appeared indistinguishable. Similarly, cocaine-induced hyperlocomotion was indifferent to blockade of newly synthesized dopamine and depletion of releasable dopamine pools. Extracellular dopamine release was similar in wild-type and triple knockout mice preexposed to the open field and given cocaine or placed immediately into the arena following injection. Since motor effects to novelty and psychostimulants depend upon frontocortical-striatal inputs, we inhibited triple knockout medial frontal cortex with GABA agonists. Locomotion was transiently increased in cocaine-injected mutants, while their supersensitive cocaine response to novelty was lost. CONCLUSIONS: These results reveal presynaptic dopamine release is not indicative of agonist-induced triple knockout hyperlocomotion. Instead, their novelty response occurs primarily through postsynaptic mechanisms and network effects.

Synapsins regulate brain-derived neurotrophic factor-mediated synaptic potentiation and axon elongation by acting on membrane rafts.

In neurons, intracellular membrane rafts are essential for specific actions of brain-derived neurotrophic factor (BDNF), which include the regulation of axon outgrowth, growth cone turning and synaptic transmission. Virtually, all the actions of BDNF are mediated by binding to its receptor, TrkB. The association of TrkB with the tyrosine kinase, Fyn, is critical for its localization to intracellular membrane rafts. Here, we show that synapsins, a family of highly amphipathic neuronal phosphoproteins, regulate membrane raft lipid composition and consequently, the ability of BDNF to regulate axon/neurite development and potentiate synaptic transmission. In the brains of mice lacking all synapsins, the expression of both BDNF and TrkB were increased, suggesting that BDNF/TrkB-mediated signaling is impaired. Consistent with this finding, synapsin-depleted neurons exhibit altered raft lipid composition, deficient targeting of Fyn to rafts, attenuated TrkB activation, and abrogation of BDNF-stimulated axon outgrowth and synaptic potentiation. Conversely, overexpression of synapsins in neuroblastoma cells results in corresponding reciprocal changes in raft lipid composition, increased localization of Fyn to rafts and promotion of BDNF-stimulated neurite formation. In the presence of synapsins, the ratio of cholesterol to estimated total phospholipids converged to 1, suggesting that synapsins act by regulating the ratio of lipids in intracellular membranes, thereby promoting lipid raft formation. These studies reveal a mechanistic link between BDNF and synapsins, impacting early development and synaptic transmission.

Telomeres, early-life stress and mental illness.

Telomeres are structures of tandem TTAGGG repeats that are found at the ends of chromosomes and preserve genomic DNA by serving as a disposable buffer to protect DNA termini during chromosome replication. In this process, the telomere itself shortens with each cell division and can consequently be thought of as a cellular 'clock', reflecting the age of a cell and the time until senescence. Telomere shortening and changes in the levels of telomerase, the enzyme that maintains telomeres, occur in the context of certain somatic diseases and in response to selected physical stressors. Emerging evidence indicates that telomeres shorten with exposure to psychosocial stress (including early-life stress) and perhaps in association with some psychiatric disorders. These discoveries suggest that telomere shortening might be a useful biomarker for the overall stress response of an organism to various pathogenic conditions. In this regard, telomeres and their response to both somatic and psychiatric illness could serve as a unifying stress-response biomarker that crosses the brain/body distinction that is often made in medicine. Prospective studies will help to clarify whether this biomarker has broad utility in psychiatry and medicine for the evaluation of responses to psychosocial stressors. The possibility that telomere shortening can be slowed or reversed by psychiatric and psychosocial interventions could represent an opportunity for developing novel preventative and therapeutic approaches.

The molecular basis of the specificity and cross-reactivity of the NeuN epitope of the neuron-specific splicing regulator, Rbfox3.

Ever since its description and the generation of its defining antibody some 20 years ago, NeuN (Neural Nuclei) has been an invaluable tool for developmental neuroscientist sand neuropathologists to identify neurons and follow their normal or malignant development [corrected].The recent identification of the splicing factor Rbfox3 as the molecule constituting the genuine NeuN epitope has opened up a novel perspective on NeuN immunostaining and its interpretation. Here, we briefly review these recent developments, and we provide a series of data that allow to rationalize the specificity of the NeuN/A60 antibody on aldehyde-fixed tissues on the one hand, and its cross-reactivity with Synapsin I and R3hdm2 on Western blots on the other. We argue that rather than being considered as a mere marker for mature neurons, Rbfox3-mediated NeuN/A60 immunoreactivity may provide a window onto neuronal biology. Specifically, we hypothesize that the phosphorylation-dependent antigenicity of the Rbfox3/NeuN epitope should allow to visualize neuronal physiology realized through Rbfox3, including splicing, on the single-cell level.

Synapsin III: role in neuronal plasticity and disease.

Synapsin III was discovered in 1998, more than two decades after the first two synapsins (synapsins I and II) were identified. Although the biology of synapsin III is not as well understood as synapsins I and II, this gene is emerging as an important factor in the regulation of the early stages of neurodevelopment and dopaminergic neurotransmission, and in certain neuropsychiatric illnesses. Molecular genetic and clinical studies of synapsin III have determined that its neurodevelopmental effects are exerted at the levels of neurogenesis and axonogenesis. In vitro voltammetry studies have shown that synapsin III can control dopamine release in the striatum. Since dopaminergic dysfunction is implicated in many neuropsychiatric conditions, one may anticipate that polymorphisms in synapsin III can exert pervasive effects, especially since it is localized to extrasynaptic sites. Indeed, mutations in this gene have been identified in individuals diagnosed with schizophrenia, bipolar disorder and multiple sclerosis. These and other findings indicate that the roles of synapsin III differ significantly from those of synapsins I and II. Here, we focus on the unique roles of the newest synapsin, and where relevant, compare and contrast these with the actions of synapsins I and II.

Transcriptome deep-sequencing and clustering of expressed isoforms from Favia corals.

BACKGROUND: Genomic and transcriptomic sequence data are essential tools for tackling ecological problems. Using an approach that combines next-generation sequencing, de novo transcriptome assembly, gene annotation and synthetic gene construction, we identify and cluster the protein families from Favia corals from the northern Red Sea. RESULTS: We obtained 80 million 75 bp paired-end cDNA reads from two Favia adult samples collected at 65 m (Fav1, Fav2) on the Illumina GA platform, and generated two de novo assemblies using ABySS and CAP3. After removing redundancy and filtering out low quality reads, our transcriptome datasets contained 58,268 (Fav1) and 62,469 (Fav2) contigs longer than 100 bp, with N50 values of 1,665 bp and 1,439 bp, respectively. Using the proteome of the sea anemone Nematostella vectensis as a reference, we were able to annotate almost 20% of each dataset using reciprocal homology searches. Homologous clustering of these annotated transcripts allowed us to divide them into 7,186 (Fav1) and 6,862 (Fav2) homologous transcript clusters (E-value ≤ 2e(-30)). Functional annotation categories were assigned to homologous clusters using the functional annotation of Nematostella vectensis. General annotation of the assembled transcripts was improved 1-3% using the Acropora digitifera proteome. In addition, we screened these transcript isoform clusters for fluorescent proteins (FPs) homologs and identified seven potential FP homologs in Fav1, and four in Fav2. These transcripts were validated as bona fide FP transcripts via robust fluorescence heterologous expression. Annotation of the assembled contigs revealed that 1.34% and 1.61% (in Fav1 and Fav2, respectively) of the total assembled contigs likely originated from the corals' algal symbiont, Symbiodinium spp. CONCLUSIONS: Here we present a study to identify the homologous transcript isoform clusters from the transcriptome of Favia corals using a far-related reference proteome. Furthermore, the symbiont-derived transcripts were isolated from the datasets and their contribution quantified. This is the first annotated transcriptome of the genus Favia, a major increase in genomics resources available in this important family of corals.

Corrigendum to "Associations of circulating cell-free DNA, C-reactive protein, and cardiometabolic risk among low-active smokers with elevated depressive symptoms" [Brain Behav. Immun. - Health 25 (2022) 100519].

[This corrects the article DOI: 10.1016/j.bbih.2022.100519.].

Synergistic interactions of repurposed drugs that inhibit Nsp1, a major virulence factor for COVID-19.

Nsp1 is one of the first proteins expressed from the SARS-CoV-2 genome and is a major virulence factor for COVID-19. A rapid multiplexed assay for detecting the action of Nsp1 was developed in cultured lung cells. The assay is based on the acute cytopathic effects induced by Nsp1. Virtual screening was used to stratify compounds that interact with two functional Nsp1 sites: the RNA-binding groove and C-terminal helix-loop-helix region. Experimental screening focused on compounds that could be readily repurposed to treat COVID-19. Multiple synergistic combinations of compounds that significantly inhibited Nsp1 action were identified. Among the most promising combinations are Ponatinib, Rilpivirine, and Montelukast, which together, reversed the toxic effects of Nsp1 to the same extent as null mutations in the Nsp1 gene.

Relationships Between Telomere Length, Plasma Glucagon-like Peptide 1, and Insulin in Early-Life Stress-Exposed Nonhuman Primates.

Background: Early-life stress is associated with alterations in telomere length, a marker of accumulated stress and aging, and a risk factor for psychiatric disorders. Nonhuman primate maternal variable foraging demand (VFD) is a validated early-life stress model, resulting in anxiety- and depressive-like symptoms in offspring. Previous studies reported increased plasma glucagon-like peptide 1 (pGLP-1) along with insulin resistance in this model. We investigated whether VFD rearing related to adult telomere length and to these neuroendocrine markers. Methods: Adult leukocyte telomere length was measured in VFD-reared (12 males, 13 females) and non-VFD-reared (9 males, 26 females) bonnet macaques. Associations between adult telomere length and adolescent fasting pGLP-1 or insulin resistance in VFD-reared versus non-VFD-reared groups were examined using regression modeling, controlling for sex, weight, and age. Results: VFD subjects had relatively longer telomeres than non-VFD subjects (p = .017), and females relatively longer than males (p = .0004). Telomere length was positively associated with pGLP-1 (p = .0009) and with reduced insulin sensitivity (p < .0001) in both sexes, but not as a function of rearing group. Conclusions: Unexpectedly, VFD was associated with longer adult telomere length. Insulin resistance may lead to higher pGLP-1 levels in adolescence, which could protect telomere length in VFD offspring as adults. Associations between adult telomere length and adolescent insulin resistance and high pGLP-1 may reflect an adaptive, compensatory response after early-life stress exposure.

Associations of circulating cell-free DNA, C-reactive protein, and cardiometabolic risk among low-active smokers with elevated depressive symptoms.

Background and aims: Cell-free DNA (cfDNA) is elevated in several disease states. Metabolic syndrome is a constellation of factors associated with poor cardiometabolic outcomes. This study examined associations of cfDNA from the nucleus (cf-nDNA) and mitochondria (cf-mtDNA), C-reactive protein (CRP), and metabolic syndrome risk, in low-active smokers with depressive symptoms. Methods: Participants (N = 109; mean age 47) self-reported medical history. Physical activity was determined by accelerometry and anthropometrics were measured. Blood was collected and analyzed for cf-nDNA, cf-mtDNA, CRP, triglycerides, high-density lipoprotein, hemoglobin A1c. A continuous metabolic syndrome composite risk score was calculated. Relationships of cf-nDNA, cf-mtDNA, CRP, and cardiometabolic risk were examined with correlations and linear regression. Results: CRP and cf-nDNA were significantly associated with metabolic syndrome risk (r = .39 and r = .31, respectively), cf-mtDNA was not (r = .01). In a linear regression, CRP and cf-nDNA significantly predicted the metabolic syndrome risk score, findings that remained significant controlling for age, gender, nicotine dependence, and physical activity. Conclusions: Associations of cf-nDNA with both CRP and metabolic risk suggest a role for cf-nDNA in inflammatory processes associated with metabolic syndrome. The negative findings for cf-mtDNA suggest distinct roles for cf-nDNA and cf-mtDNA in these processes.

Novel internal regions of fluorescent proteins undergo divergent evolutionary patterns.

Over the past decade, fluorescent proteins (FPs) have become ubiquitous tools in biological research. Yet, little is known about the natural function or evolution of this superfamily of proteins that originate from marine organisms. Using molecular phylogenetic analyses of 102 naturally occurring cyan fluorescent proteins, green fluorescent proteins, red fluorescent proteins, as well as the nonfluorescent (purple-blue) protein sequences (including new FPs from Lizard Island, Australia) derived from organisms with known geographic origin, we show that FPs consist of two distinct and novel regions that have evolved under opposite and sharply divergent evolutionary pressures. A central region is highly conserved, and although it contains the residues that form the chromophore, its evolution does not track with fluorescent color and evolves independently from the rest of the protein. By contrast, the regions enclosing this central region are under strong positive selection pressure to vary its sequence and yet segregate well with fluorescence color emission. We did not find a significant correlation between geographic location of the organism from which the FP was isolated and molecular evolution of the protein. These results define for the first time two distinct regions based on evolution for this highly compact protein. The findings have implications for more sophisticated bioengineering of this molecule as well as studies directed toward understanding the natural function of FPs.

Molecular markers of neuroendocrine function and mitochondrial biogenesis associated with early life stress.

OBJECTIVE: Glucocorticoid receptor gene (NR3C1) promoter methylation influences cellular expression of the glucocorticoid receptor and is a proposed mechanism by which early life stress impacts neuroendocrine function. Mitochondria are sensitive and responsive to neuroendocrine stress signaling through the glucocorticoid receptor, and recent evidence with this sample and others shows that mitochondrial DNA copy number (mtDNAcn) is increased in adults with a history of early stress. No prior work has examined the role of NR3C1 methylation in the association between early life stress and mtDNAcn alterations. METHODS: Adult participants (n = 290) completed diagnostic interviews and questionnaires characterizing early stress and lifetime psychiatric symptoms. Medical conditions, active substance abuse, and prescription medications other than oral contraceptives were exclusionary. Subjects with a history of lifetime bipolar, obsessive-compulsive, or psychotic disorders were excluded; individuals with other forms of major psychopathology were included. Whole blood mtDNAcn was measured using qPCR; NR3C1 methylation was measured via pyrosequencing. Multiple regression and bootstrapping procedures tested NR3C1 methylation as a mediator of effects of early stress on mtDNAcn. RESULTS: The positive association between early adversity and mtDNAcn (p = .02) was mediated by negative associations of early adversity with NR3C1 methylation (p = .02) and NR3C1 methylation with mtDNAcn (p < .001). The indirect effect involving early adversity, NR3C1 methylation, and mtDNAcn was significant (95 % CI [.002, .030]). CONCLUSIONS: NR3C1 methylation significantly mediates the association between early stress and mtDNAcn, suggesting that glucocorticoid receptor signaling may be a mechanistic pathway underlying mtDNAcn alterations of interest for future longitudinal work.

Childhood maltreatment, behavioral adjustment, and molecular markers of cellular aging in preschool-aged children: A cohort study.

OBJECTIVE: Childhood maltreatment is a major risk factor for the development of behavioral problems and poor physical and mental health. Accelerated cellular aging, through reduced telomere length and mitochondrial dysfunction, may be a mechanism underlying these associations. METHODS: Families with (n = 133) and without (n = 123) child welfare documentation of moderate-severe maltreatment in the past six months participated in this study. Children ranged in age from 3 to 5 years, were racially and ethnically diverse, and 91% qualified for public assistance. Structured record review and interviews were used to assess a history of maltreatment and other adversities. Telomere length and mitochondrial DNA copy number (mtDNAcn) were measured from saliva DNA using real-time PCR. Measures were repeated at a six-month follow-up assessment. Repeated measures general linear models were used to examine the effects of maltreatment and other adversities on telomere length and mtDNAcn over time. RESULTS: Maltreatment and other adverse experiences were significant positive predictors of both telomere length and mtDNAcn over time. Internalizing and externalizing behavior problems were also both significantly associated with telomere length, but only internalizing symptoms were associated with mtDNAcn. CONCLUSIONS: This is the first study to show that mtDNAcn is altered in children with stress and trauma, and the findings are consistent with recent studies of adults. Surprisingly, children who experienced moderate-severe levels of maltreatment in the prior six months had longer telomeres, possibly reflecting compensatory changes in response to recent trauma. Telomere length and mtDNAcn were also associated with behavioral problems, suggesting that these measures of cellular aging may be causally implicated in the pathophysiology of stress-related conditions.

Three-dimensional architecture of presynaptic terminal cytomatrix.

Presynaptic terminals are specialized for mediating rapid fusion of synaptic vesicles (SVs) after calcium influx. The regulated trafficking of SVs likely results from a highly organized cytomatrix. How this cytomatrix links SVs, maintains them near the active zones (AZs) of release, and organizes docked SVs at the release sites is not fully understood. To analyze the three-dimensional (3D) architecture of the presynaptic cytomatrix, electron tomography of presynaptic terminals contacting spines was performed in the stratum radiatum of the rat hippocampal CA1 area. To preserve the cytomatrix, hippocampal slices were immobilized using high-pressure freezing, followed by cryosubstitution and embedding. SVs are surrounded by a dense network of filaments. A given vesicle is connected to approximately 1.5 neighboring ones. SVs at the periphery of this network are also linked to the plasma membrane, by longer filaments. More of these filaments are found at the AZ. At the AZ, docked SVs are grouped around presynaptic densities. Filaments with adjacent SVs emerge from these densities. Immunogold localizations revealed that synapsin is located in the presynaptic bouton, whereas Bassoon and CAST (ERC2) are at focal points next to the AZ. In synapsin triple knock-out mice, the number of SVs is reduced by 63%, but the size of the boutons is reduced by only 18%, and the mean distance of SVs to the AZ is unchanged. This 3D analysis reveals the morphological constraints exerted by the presynaptic molecular scaffold. SVs are tightly interconnected in the axonal bouton, and this network is preferentially connected to the AZ.

Telomerase levels in schizophrenia: a preliminary study.

We previously demonstrated that telomere length was markedly reduced in peripheral blood lymphocytes from individuals with schizophrenia. Since reduced telomere length can be caused by decreased telomerase activity, we quantitated basal telomerase activity in peripheral blood lymphocytes derived from individuals with schizophrenia (n=53), unaffected relatives (n=31) and unrelated controls (n=59). Telomerase activity varied greatly among individuals, suggesting that this enzymatic activity is affected by various factors. We observed a nominally significant decrease in telomerase activity among individuals with schizophrenia compared to unaffected individuals (unaffected relatives and unrelated controls). Further studies are needed to investigate the role of telomerase in schizophrenia.

Strict regulation of gene expression from a high-copy plasmid utilizing a dual vector system.

High-copy plasmids are useful for producing large quantities of plasmid DNA, but are generally inadequate for tightly regulating gene expression. Attempts to suppress expression of genes on high-copy plasmids often results in residual or "leaky" production of protein. For stringent regulation of gene expression, it is often necessary to excise the gene of interest and subclone it into a low-copy plasmid. Here, we report a dual plasmid technique that enables tight regulation of gene expression driven by the lac promoter in a high-copy vector. A series of plasmids with varying copies of the lacI(q) gene have been constructed to permit titration of the LacI protein. When a high-copy plasmid is transformed along with the appropriate lacI(q)-containing plasmid, tight gene regulation is achieved, thus eliminating the need to subclone genes into low-copy plasmids. In addition, we show that this dual plasmid technique enables high-copy gene expression of a protein lethal to Escherichia coli, the ccdB protein. In principle, this technique can be applied to any high-copy plasmid containing the popular pUC replication of origin and provides an easier means of obtaining rigid control over gene expression.

Patterns of fluorescent protein expression in Scleractinian corals.

Biofluorescence exists in only a few classes of organisms, with Anthozoa possessing the majority of species known to express fluorescent proteins. Most species within the Anthozoan subgroup Scleractinia (reef-building corals) not only express green fluorescent proteins, they also localize the proteins in distinct anatomical patterns.We examined the distribution of biofluorescence in 33 coral species, representing 8 families, from study sites on Australia's Great Barrier Reef. For 28 of these species, we report the presence of biofluorescence for the first time. The dominant fluorescent emissions observed were green (480-520 nm) and red (580-600 nm). Fluorescent proteins were expressed in three distinct patterns (highlighted, uniform, and complementary) among specific anatomical structures of corals across a variety of families. We report no significant overlap between the distribution of fluorescent proteins and the distribution of zooxanthellae. Analysis of the patterns of fluorescent protein distribution provides evidence that the scheme in which fluorescent proteins are distributed among the anatomical structures of corals is nonrandom. This targeted expression of fluorescent proteins in corals produces contrast and may function as a signaling mechanism to organisms with sensitivity to specific wavelengths of light.

A protein kinase A-dependent molecular switch in synapsins regulates neurite outgrowth.

Cyclic AMP (cAMP) promotes neurite outgrowth in a variety of neuronal cell lines through the activation of protein kinase A (PKA). We show here, using both Xenopus laevis embryonic neuronal culture and intact X. laevis embryos, that the nerve growth-promoting action of cAMP/PKA is mediated in part by the phosphorylation of synapsins at a single amino acid residue. Expression of a mutated form of synapsin that prevents phosphorylation at this site, or introduction of phospho-specific antibodies directed against this site, decreased basal and dibutyryl cAMP-stimulated neurite outgrowth. Expression of a mutation mimicking constitutive phosphorylation at this site increased neurite outgrowth, both under basal conditions and in the presence of a PKA inhibitor. These results provide a potential molecular approach for stimulating neuron regeneration, after injury and in neurodegenerative diseases.