Specification of Neuronal Polarity Regulated by Local Translation of CRMP2 and Tau via the mTOR-p70S6K Pathway

Mammalian target of rapamycin (mTOR) is an important regulator of neuronal development and functions. Although it was reported recently that mTOR signaling is critical for neuronal polarity, the underlying mechanism remains unclear. Here, we describe the molecular pathway of mTOR-dependent axon specification, in which the collapsing response mediator protein 2 (CRMP2) and Tau are major downstream targets. The activity of mTOR effector 70-kDa ribosomal protein S6 kinase (p70S6K) specifically increases in the axon during neuronal polarity formation. The mTOR inhibitor rapamycin suppresses the translation of some neuronal polarity proteins, including CRMP2 and Tau, thereby inhibiting axon formation. In contrast, constitutively active p70S6K up-regulates the translation of these molecules, thus inducing multiple axons. Exogenous CRMP2 and Tau facilitate axon formation, even in the presence of rapamycin. In the 5′-untranslated region of Tau and CRMP2 mRNAs, we identified a 5′-terminal oligopyrimidine tract, which mediates mTOR-governed protein synthesis. The 5′-terminal oligopyrimidine tract sequences of CRMP2 and Tau mRNAs strongly contribute to the up-regulation of their translation in the axon in response to the axonal activation of the mTOR-p70S6K pathway. Taken together, we conclude that the local translation of CRMP2 and Tau, regulated by mTOR-p70S6K, is critical for the specification of neuronal polarity.To function, developing neurons must establish axonal-dendritic polarity. The polarization processes have been well studied in cultured hippocampal neurons (1, 2). They first form lamellipodia and small protrusion veils shortly after plating (stage 1) and then extend several immature neurites within 12–24 h (stage 2). One of the neurites rapidly elongates and becomes the axon, whereas the others become dendrites (stage 3). During these processes, a number of molecules coordinately control the axon specification (3). These molecules spatially regulate cytoskeletal networks of actin filaments and microtubules for specification and maintenance of a single axon.Mammalian target of rapamycin (mTOR)² is a serine/threonine protein kinase and mainly controls protein synthesis via phosphorylation of its downstream targets, such as eukaryotic translation initiation factor 4E (eIF-4E)-binding protein 1 (4E-BP1) and 70-kDa ribosomal S6 protein kinase (p70S6K) (4). In the nervous system, mTOR plays important roles in neuronal survival, dendritic arbor formation, synaptic plasticity, learning, and memory (5). Notably, in the regulation of synaptic plasticity, a number of mRNAs are locally translated via activation of the mTOR pathway in response to extracellular signals (5–7). Recently, two research groups reported that the mTOR pathway is also critical for the formation of neuronal polarity (8, 9). They proposed that the mTOR pathway controls the synthesis of polarity proteins, such as synapses of the amphid-detective (SAD) kinases (8) and Rap1 (9), and therefore regulates the neuronal polarity. However, the underlying molecular mechanism remains to be fully understood. Here, we demonstrate that p70S6K is necessary and sufficient for the mTOR-controlled axon formation as a downstream effector of mTOR. The mTOR-p70S6K pathway regulates translation of several neuronal polarity genes, including SAD kinases and Rap1. In particular, the expressions of collapsing response mediator protein 2 (CRMP2) and microtubule-associated protein Tau are highly dependent on mTOR-governed protein synthesis. We further identified a 5′-terminal oligopyrimidine (5′-TOP) tract, which mediates mTOR-governed protein synthesis (10), in the 5′-UTRs of CRMP2 and Tau mRNAs. In the axon of developing neurons, the translation of CRMP2 and Tau mRNAs is spatially controlled by the mTOR-p70S6K pathway via the 5′-TOP sequences, resulting in accumulation of CRMP2 and Tau proteins in the axon. Thus, our study substantially improves understanding of molecular mechanisms underlying the mTOR-dependent development of neuronal polarity.     

Collapsin Response Mediator Protein 5 (CRMP5) Induces Mitophagy,Thereby Regulating Mitochondrion Numbers in Dendrites

Degradation of damaged mitochondria by mitophagy is an essential process to ensure cell homeostasis. Because neurons, which have a high energy demand, are particularly dependent on the mitochondrial dynamics, mitophagy represents a key mechanism to ensure correct neuronal function. Collapsin response mediator proteins 5 (CRMP5) belongs to a family of cytosolic proteins involved in axon guidance and neurite outgrowth signaling during neural development. CRMP5, which is highly expressed during brain development, plays an important role in the regulation of neuronal polarity by inhibiting dendrite outgrowth at early developmental stages. Here, we demonstrated that CRMP5 was present in vivo in brain mitochondria and is targeted to the inner mitochondrial membrane. The mitochondrial localization of CRMP5 induced mitophagy. CRMP5 overexpression triggered a drastic change in mitochondrial morphology, increased the number of lysosomes and double membrane vesicles termed autophagosomes, and enhanced the occurrence of microtubule-associated protein 1 light chain 3 (LC3) at the mitochondrial level. Moreover, the lipidated form of LC3, LC3-II, which triggers autophagy by insertion into autophagosomes, enhanced mitophagy initiation. Lysosomal marker translocates at the mitochondrial level, suggesting autophagosome-lysosome fusion, and induced the reduction of mitochondrial content via lysosomal degradation. We show that during early developmental stages the strong expression of endogenous CRMP5, which inhibits dendrite growth, correlated with a decrease of mitochondrial content. In contrast, the knockdown or a decrease of CRMP5 expression at later stages enhanced mitochondrion numbers in cultured neurons, suggesting that CRMP5 modulated these numbers. Our study elucidates a novel regulatory mechanism that utilizes CRMP5-induced mitophagy to orchestrate proper dendrite outgrowth and neuronal function.     

Phosphatidylserine-dependent neuroprotective signaling promoted by docosahexaenoic acid

Enrichment of polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA, 22:6n?3), in the brain is known to be critical for optimal brain development and function. Mechanisms for DHA’s beneficial effects in the nervous system are not clearly understood at present. DHA is incorporated into the phospholipids in neuronal membranes, which in turn can influence not only the membrane chemical and physical properties but also the cell signaling involved in neuronal survival, proliferation and differentiation. Our studies have indicated that DHA supplementation promotes phosphatidylserine (PS) accumulation and inhibits neuronal cell death under challenged conditions, supporting a notion that DHA is an important neuroprotective agent. This article summarizes our findings on the DHA-mediated membrane-related signaling mechanisms that might explain some of the beneficial effects of DHA, particularly on neuronal survival.     

Identification of a Dithiol-disulfide Switch in Collapsin Response Mediator Protein 2 (CRMP2) That Is Toggled in a Model of Neuronal Differentiation

Vertebrate-specific glutaredoxin 2 (Grx2) is expressed in at least two isoforms, mitochondrial Grx2a and cytosolic Grx2c. We have previously shown that cytosolic Grx2 is essential for embryonic development of the brain. In particular, we identified collapsin response mediator protein 2 (CRMP2/DPYSL2), a mediator of the semaphorin-plexin signaling pathway, as redox-regulated target of Grx2c and demonstrated that this regulation is required for normal axonal outgrowth. In this study, we demonstrate the molecular mechanism of this regulation, a specific and reversible intermolecular Cys-504-Cys-504 dithiol-disulfide switch in homotetrameric CRMP2. This switch determines two conformations of the quaternary CRMP2 complex that controls axonal outgrowth and thus neuronal development.     

CRMP5 regulates generation and survival of newborn neurons in olfactory and hippocampal neurogenic areas of the adult mouse brain

The Collapsin Response Mediator Proteins (CRMPS) are highly expressed in the developing brain, and in adult brain areas that retain neurogenesis, ie: the olfactory bulb (OB) and the dentate gyrus (DG). During brain development, CRMPs are essentially involved in signaling of axon guidance and neurite outgrowth, but their functions in the adult brain remain largely unknown. CRMP5 has been initially identified as the target of auto-antibodies involved in paraneoplasic neurological diseases and further implicated in a neurite outgrowth inhibition mediated by tubulin binding. Interestingly, CRMP5 is also highly expressed in adult brain neurogenic areas where its functions have not yet been elucidated. Here we observed in both neurogenic areas of the adult mouse brain that CRMP5 was present in proliferating and post-mitotic neuroblasts, while they migrate and differentiate into mature neurons. In CRMP5(-/-) mice, the lack of CRMP5 resulted in a significant increase of proliferation and neurogenesis, but also in an excess of apoptotic death of granule cells in the OB and DG. These findings provide the first evidence that CRMP5 is involved in the generation and survival of newly generated neurons in areas of the adult brain with a high level of activity-dependent neuronal plasticity.     

Dopamine and α-synuclein dysfunction in Smad3 null mice

Background

Recent studies suggest that the pathogenic process in neurodegenerative disorders may disrupt mature neuronal circuitries and neurogenesis in the adult brain. Abnormal activation of CDK5 is associated with neurodegenerative disorders, and recently a critical role for CDK5 in adult neurogenesis has been identified. We have developed an in vitro model of abnormal CDK5 activation during adult hippocampal neurogenesis, and here we used this model to investigate aberrantly phosphorylated downstream targets of CDK5.

Results

Abnormal CDK5 activation in an in vitro model of adult neurogenesis results in hyperphosphorylation of collapsin-response mediator protein-2 (CRMP2) and impaired neurite outgrowth. Inhibition of CDK5, or expression of a non-phosphorylatable (S522A) CRMP2 construct reduced CRMP2 hyperphosphorylation, and reversed neurite outgrowth deficits. CRMP2 plays a role in microtubule dynamics; therefore we examined the integrity of microtubules in this model using biochemical and electron microscopy techniques. We found that microtubule organization was disrupted under conditions of CDK5 activation. Finally, to study the relevance of these findings to neurogenesis in neurodegenerative conditions associated with HIV infection, we performed immunochemical analyses of the brains of patients with HIV and transgenic mice expressing HIV-gp120 protein. CDK5-mediated CRMP2 phosphorylation was significantly increased in the hippocampus of patients with HIV encephalitis and in gp120 transgenic mice, and this effect was rescued by genetic down-modulation of CDK5 in the mouse model.

Conclusions

These results reveal a functional mechanism involving microtubule destabilization through which abnormal CDK5 activation and CRMP2 hyperphosphorylation might contribute to defective neurogenesis in neurodegenerative disorders such as HIV encephalitis.     

Glycogen Synthase Kinase 3β Orchestrates Microtubule Remodeling in Compensatory Glomerular Adaptation to Podocyte Depletion

Reminiscent of neural repair, following podocyte depletion, remnant-surviving podocytes exhibit a considerable adaptive capacity to expand and cover the denuded renal glomerular basement membrane. Microtubules, one of the principal cytoskeletal components of podocyte major processes, play a crucial role in podocyte morphogenesis and podocyte process outgrowth, branching, and elongation. Here, we demonstrated that the microtubule-associated proteins Tau and collapsin response mediator protein (CRMP) 2, key regulators of microtubule dynamics, were abundantly expressed by glomerular podocytes in vivo and in vitro, interacted with glycogen synthase kinase (GSK)3β, and served as its putative substrates. GSK3β overactivity induced by adriamycin injury or by a constitutively active mutant of GSK3β augmented phosphorylation of Tau and CRMP2, concomitant with microtubule depolymerization, cell body shrinkage, and shortening of podocyte processes. Conversely, inhibition of GSK3β by a dominant negative mutant or by lithium, a Food and Drug Administration-approved neuroprotective mood stabilizer, diminished Tau and CRMP2 phosphorylation, resulting in microtubule polymerization, podocyte expansion, and lengthening of podocyte processes. In a mouse model of adriamycin-induced podocyte depletion and nephropathy, delayed administration of a single low dose of lithium attenuated proteinuria and ameliorated progressive glomerulosclerosis despite no correction of podocytopenia. Mechanistically, lithium therapy obliterated GSK3β overactivity, mitigated phosphorylation of Tau and CRMP2, and enhanced microtubule polymerization and stabilization in glomeruli in adriamycin-injured kidneys, associated with elongation of podocyte major processes. Collectively, our findings suggest that the GSK3β-dictated podocyte microtubule dynamics might serve as a novel therapeutic target to reinforce the compensatory glomerular adaptation to podocyte loss.     

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.     

Neuroprotection against traumatic brain injury by a peptide derived from the collapsin response mediator protein 2 (CRMP2)

Neurological disabilities following traumatic brain injury (TBI) may be due to excitotoxic neuronal loss. The excitotoxic loss of neurons following TBI occurs largely due to hyperactivation of N-methyl-d-aspartate receptors (NMDARs), leading to toxic levels of intracellular Ca(2+). The axon guidance and outgrowth protein collapsin response mediator protein 2 (CRMP2) has been linked to NMDAR trafficking and may be involved in neuronal survival following excitotoxicity. Lentivirus-mediated CRMP2 knockdown or treatment with a CRMP2 peptide fused to HIV TAT protein (TAT-CBD3) blocked neuronal death following glutamate exposure probably via blunting toxicity from delayed calcium deregulation. Application of TAT-CBD3 attenuated postsynaptic NMDAR-mediated currents in cortical slices. In exploring modulation of NMDARs by TAT-CBD3, we found that TAT-CBD3 induced NR2B internalization in dendritic spines without altering somal NR2B surface expression. Furthermore, TAT-CBD3 reduced NMDA-mediated Ca(2+) influx and currents in cultured neurons. Systemic administration of TAT-CBD3 following a controlled cortical impact model of TBI decreased hippocampal neuronal death. These findings support TAT-CBD3 as a novel neuroprotective agent that may increase neuronal survival following injury by reducing surface expression of dendritic NR2B receptors.     

mTOR controls FLIPS translation and TRAIL sensitivity in glioblastoma multiforme cells

The tumor-selective, proapoptotic, death receptor ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a mediator of antitumor drug activity and in itself is a promising agent for the treatment of human malignancies. Like many tumors, however, glioblastoma multiforme (GBM), the most fatal form of glioma, exhibits a range of TRAIL sensitivity, and only a small percentage of GBM tumors undergo TRAIL-induced apoptosis. We here show that TRAIL resistance in GBM is a consequence of overexpression of the short isoform of the caspase-8 inhibitor, c-FLICE inhibitory protein (FLIP(S)), and that FLIP(S) expression is in turn translationally enhanced by activation of the Akt-mammalian target of rapamycin (mTOR)-p70 S6 kinase 1 (S6K1) pathway. Conversely, pharmacologic or genetic inhibition of mTOR, or the mTOR target S6K1, suppresses polyribosomal accumulation of FLIP(S) mRNA, FLIP(S) protein expression, and TRAIL resistance. In archived material from 12 human GBM tumors, PTEN status was a predictor of activation of the Akt-mTOR-S6K1 pathway and of FLIP(S) levels, while in xenografted human GBM, activation status of the PTEN-Akt-mTOR pathway distinguished the tumors inherently sensitive to TRAIL from those which could be sensitized by the mTOR inhibitor rapamycin. These results define the mTOR pathway as a key limiter of tumor elimination by TRAIL-mediated mechanisms, provide a means by which the TRAIL-sensitive subset of GBM can be identified, and provide rationale for the combined use of TRAIL with mTOR inhibitors in the treatment of human cancers.     

Effects of Hypothyroidism on Expression of CRMP2B and ARPC5 during Development of the Rat Frontal Cortex

Congenital hypothyroidism (CH) can lead to irreversible central nervous system (CNS) damage. However, the pathogenesis of the developmental brain disorders caused by CH has not been completely elucidated. ARPC5 and CRMP2 are closely associated with neurite outgrowth in brain development. Thus, the aim of the present study was to determine whether CRMP2B and ARPC5 expression is altered in the developing cerebral cortex of rats with CH. Control rats and rats with hypothyroidism were sacrificed at birth and at 15 days postpartum. We performed qRT-PCR to detect differences in the crmp2B and arpc5 mRNA expression in the right half of the frontal cortex of these rats. Western blotting was then used to detect differences in CRMP2B and ARPC5 protein expression. Furthermore, immunohistochemical analysis was performed on the left half of the frontal cortex to detect abnormal localization of CRMP2B and ARPC5. Results showed increased expression of the nuclear short isoform of CRMP2B and decreased expression of full-length CRMP2B and ARPC5 in cortical neurons of rats with hypothyroidism. These findings demonstrate that reduced levels of thyroid hormones can inhibit the expression of full-length CRMP2B and ARPC5 and promote nuclear transformation of the short isoform of CRMP2B. CRMP2B and ARPC5 may participate in CNS injury mediated by hypothyroidism by inducing neurite outgrowth inhibition and cytoskeletal protein disorganization.     

Thyroid hormone stimulates protein synthesis in the cardiomyocyte by activating the Akt-mTOR and p70S6K pathways

Thyroid hormones affect cardiac growth and phenotype; however, the mechanisms by which the hormones induce cardiomyocyte hypertrophy remain uncharacterized. Tri-iodo-L-thyronine (T3) treatment of cultured cardiomyocytes for 24 h resulted in a 41 +/- 5% (p < 0.001) increase in [(3)H]leucine incorporation into total cellular protein. This response was abrogated by the phosphatidylinositol 3-kinase (PI3K) inhibitor, wortmannin. Co-immunoprecipitation studies showed a direct interaction of cytosol-localized thyroid hormone receptor TRalpha1 and the p85alpha subunit of PI3K. T3 treatment rapidly increased PI3K activity by 52 +/- 3% (p < 0.005), which resulted in increased phosphorylation of downstream kinases Akt and mammalian target of rapamycin (mTOR). This effect was abrogated by pretreatment with wortmannin or LY294002. Phosphorylation of p70(S6K), a known target of mTOR, occurred rapidly following T3 treatment and was inhibited by rapamycin and wortmannin. In contrast, phosphorylation of the p85 variant of S6K in response to T3 was not blocked by LY294002, wortmannin, or rapamycin, thus supporting a T3-activated pathway independent of PI3K and mTOR. 40 S ribosomal protein S6, a target of p70(S6K), and 4E-BP1, a target of mTOR, were both phosphorylated within 15-25 min of T3 treatment and could be inhibited by wortmannin and rapamycin. Thus, rapid T3-mediated activation of PI3K by cytosolic TRalpha1 and subsequent activation of the Akt-mTOR-S6K signaling pathway may underlie one of the mechanisms by which thyroid hormone regulates physiological cardiac growth.     

Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.     

Mechanisms of TSC-mediated control of synapse assembly and axon guidance

Tuberous sclerosis complex is a dominant genetic disorder produced by mutations in either of two tumor suppressor genes, TSC1 and TSC2; it is characterized by hamartomatous tumors, and is associated with severe neurological and behavioral disturbances. Mutations in TSC1 or TSC2 deregulate a conserved growth control pathway that includes Ras homolog enriched in brain (Rheb) and Target of Rapamycin (TOR). To understand the function of this pathway in neural development, we have examined the contributions of multiple components of this pathway in both neuromuscular junction assembly and photoreceptor axon guidance in Drosophila. Expression of Rheb in the motoneuron, but not the muscle of the larval neuromuscular junction produced synaptic overgrowth and enhanced synaptic function, while reductions in Rheb function compromised synapse development. Synapse growth produced by Rheb is insensitive to rapamycin, an inhibitor of Tor complex 1, and requires wishful thinking, a bone morphogenetic protein receptor critical for functional synapse expansion. In the visual system, loss of Tsc1 in the developing retina disrupted axon guidance independently of cellular growth. Inhibiting Tor complex 1 with rapamycin or eliminating the Tor complex 1 effector, S6 kinase (S6k), did not rescue axon guidance abnormalities of Tsc1 mosaics, while reductions in Tor function suppressed those phenotypes. These findings show that Tsc-mediated control of axon guidance and synapse assembly occurs via growth-independent signaling mechanisms, and suggest that Tor complex 2, a regulator of actin organization, is critical in these aspects of neuronal development.     

Proteomic analysis of neonatal mouse brain: evidence for hypoxia- and ischemia-induced dephosphorylation of collapsin response mediator proteins

Perinatal hypoxia and ischemia (HI) are a significant cause of mortality and morbidity. To understand the molecular mechanisms for HI-induced brain damage, here we used a proteomic approach to analyze the alteration and modification of proteins in neonatal mouse brain 24 h after HI treatment. Significant changes of collapsin response mediator proteins (CRMPs) were observed in HI brain. CRMPs are a family of cytosolic proteins involved in axonal guidance and neuronal outgrowth. We found that CRMP2, CRMP4 and CRMP5 proteins were altered post-translationally after HI treatment. Mass spectrometric and Western blot analyses detected hypophosphorylated CRMP proteins after HI. Further analysis of CRMP kinases indicated inactivation of cyclin dependent kinase 5 (CDK5), a priming kinase of CRMPs and a neuronal specific kinase that plays pivotal roles in neuronal development and survival. The reduction of CDK5 activity was associated with underexpression of its activator p35. Taken together, our findings reveal HI-induced dephosphorylation of CRMPs in neonatal brain and suggest a novel mechanism for this modification. Hypophosphorylated CRMPs might be implicated in the pathogenesis of HI-related neurological disorders.     

Nutritional n-3 polyunsaturated fatty acids deficiency alters cannabinoid receptor signaling pathway in the brain and associated anxiety-like behavior in mice

N-3 polyunsaturated fatty acids (PUFAs) cannot be synthesized de novo in mammals and need to be provided by dietary means. In the brain, the main n-3 PUFA is docosahexaenoic acid (DHA), which is a key component of neuronal membranes. A low dietary level of DHA has been associated with increased risk of developing neuropsychiatric diseases; however, the mechanisms involved remain to be determined. In this study, we found that long-term exposure to an n-3 deficient diet decreases the level of DHA in the brain and impairs the cannabinoid receptor signaling pathway in mood-controlling structures. In n-3 deficient mice, the effect of the cannabinoid agonist WIN55,212-2 in an anxiety-like behavior test was abolished. In addition, the cannabinoid receptor signaling pathways were altered in the prefrontal cortex and the hypothalamus. Consequently, our data suggest that behavioral changes linked to an n-3 dietary deficiency are due to an alteration in the endocannabinoid system in specific brain areas.     

SUMOylation alters CRMP2 regulation of calcium influx in sensory neurons

The axon/dendrite specification collapsin response mediator protein 2 (CRMP2) bidirectionally modulates N-type voltage-gated Ca²⁺ channels (CaV2.2). Here we demonstrate that small ubiquitin-like modifier (SUMO) protein modifies CRMP2 via the SUMO E2-conjugating enzyme Ubc9 in vivo. Removal of a SUMO conjugation site KMD in CRMP2 (K374A/M375A/D376A; CRMP2_AAA) resulted in loss of SUMOylated CRMP2 without compromising neurite branching, a canonical hallmark of CRMP2 function. Increasing SUMOylation levels correlated inversely with calcium influx in sensory neurons. CRMP2 deSUMOylation by SUMO proteases SENP1 and SENP2 normalized calcium influx to those in the CRMP2_AAA mutant. Thus, our results identify a novel role for SUMO modification in CRMP2/CaV2.2 signaling pathway.