Superior Cervical Ganglion-10 Research Articles

The Interaction between ADK and SCG10 Regulate the Repair of Nerve Damage

The cytoskeleton must be remodeled during neurite outgrowth, and Superior Cervical Ganglion 10 (SCG10) plays a critical role in this process by depolymerizing Microtubules (MTs), conferring highly dynamic properties to the MTs. However, the precise mechanism of action of SCG10 in the repair of injured neurons remains largely uncertain. Using transcriptomic identification, we discovered that SCG10 expression was downregulated in neurons after Spinal Cord Injury (SCI). Additionally, through mass spectrometry identification, immunoprecipitation, and pull-down assays, we established that SCG10 could interact with Adenosine Kinase (ADK). Furthermore, we developed an excitotoxicity-induced neural injury model and discovered that ADK suppressed injured neurite re-growth, whereas, through overexpression and small molecule interference experiments, SCG10 enhanced it. Moreover, we discovered ADK to be the upstream of SCG10. More importantly, the application of the ADK inhibitor called 5-Iodotubercidin (5-ITu) was found to significantly enhance the recovery of motor function in mice with SCI. Consequently, our findings suggest that ADK plays a negative regulatory role in the repair of injured neurons. Herein, we propose a molecular interaction model of the SCG10-ADK axis to regulate neuronal recovery.

SCG10 is required for peripheral axon maintenance and regeneration in mice.

Proper microtubule dynamics is critical for neuronal morphogenesis and functions, and its dysregulation results in neurological disorders and regeneration failure. Superior cervical ganglion-10 (SCG10, also known as Stathmin-2) is a well-known regulator of microtubule dynamics in neurons, but its functions in the peripheral nervous system (PNS) remain largely unknown. Here, we show that Scg10 knockout mice exhibit severely progressive motor and sensory dysfunctions with significant sciatic nerve myelination deficits and neuromuscular degeneration. Furthermore, increased microtubule stability, shown by a significant increase in tubulin acetylation and decrease in tubulin tyrosination, and decreased axonal transport occur in Scg10 knockout DRG neurons. Meanwhile, SCG10 depletion impairs axon regeneration in both injured mouse sciatic nerve and cultured DRG neurons following replating, and the impaired axon regeneration was found to be induced by a lack of SCG10-mediated microtubule dynamics in the neurons. Thus, our results highlight the importance of SCG10 in peripheral axon maintenance and regeneration.

Regenerative plasticity of intact human skin axons

The evaluation of human epidermal innervation and its impact by disease has largely focused on rigorous immunohistochemical counts of PGP 9.5 labelled axons. In this brief and preliminary report, we expand the repertoire of epidermal axon markers to include those with an influence on their regenerative plasticity. We studied human lower limb punch skin samples with tandem analyses of their mRNA content using qRT-PCR. Normal human subjects (n = 11) and two patients with newly diagnosed CIDP were sampled with the latter undergoing serial tandem biopsies before and after 3 months of immunotherapy. Controls expressed regeneration proteins within dermal and epidermal axons: GAP43 (growth associated protein 43), Shh (sonic hedgehog) and SCG (superior cervical ganglion-10; stathmin 2). Moreover, this expression accompanied intraepidermal nerve fiber density (IENF) within normal established values. CIDP patients had lower IENF but also expressed GAP43, Shh, and SCG. Tandem qRT-PCR identified confirmed the presence not only of these plasticity markers but of additional regeneration related mRNAs. CIDP patients had marked elevation of several mRNAs, with improvement following treatment. The findings support the concept of dynamic skin axon plasticity in humans is relevant toward consideration of newer therapeutic approaches.

Gait Deficits and Loss of Striatal Tyrosine Hydroxlase/Trk-B are Restored Following 7,8-Dihydroxyflavone Treatment in a Progressive MPTP Mouse Model of Parkinson’s Disease

Parkinson’s disease (PD) is caused by neurodegeneration of nigrostriatal neurons, resulting in dopamine (DA) stimulated motor deficits. Like brain derived neurotrophic factor (BDNF), 7,8-dihydroxyflavone (DHF) is an agonist of the tropomyosin receptor kinase-B (TrkB) and stimulates the same secondary cascades that promote neuronal growth, survival and differentiation. We used our progressive mouse model of PD by administering increasing doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over 4 weeks (5 days/week), and then treated mice with DHF for 4 weeks after the cessation of the toxin injections (i.e., restoration). Mice treated with DHF recovered motorically, even after MPTP administration. Despite a 75% loss of tyrosine hydroxylase (TH) expression in the dorsolateral (DL) striatum in the MPTP group, mice treated with DHF had a recovery comparable to that found in the respective control. There was no recovery of DA tissue levels within the DL striatum. In both the DL striatum and substantia nigra (SN)/midbrain, phosphorylated TrkB and secondary messengers were significantly increased following DHF compared to the MPTP only group. Expression of the sprouting biomarker, superior cervical ganglion 10 (SCG10), was increased ∼20% in the DL striatum and 66% in the SN/midbrain in mice treated with DHF compared to the MPTP only group. We report that after 4 weeks of progressive MPTP administration, DHF can restore motor deficits and TH within the DL striatum in a TrkB-dependent manner. Our data suggests that DHF may help alleviate motor symptoms of PD and restore the loss of DA terminals within the striatum.

A Conditioning Sciatic Nerve Lesion Triggers a Pro-regenerative State in Primary Sensory Neurons Also of Dorsal Root Ganglia Non-associated With the Damaged Nerve.

The primary sensory neurons of dorsal root ganglia (DRG) are a very useful model to study the neuronal regenerative program that is a prerequisite for successful axon regeneration after peripheral nerve injury. Seven days after a unilateral sciatic nerve injury by compression or transection, we detected a bilateral increase in growth-associated protein-43 (GAP-43) and superior cervical ganglion-10 (SCG-10) mRNA and protein levels not only in DRG neurons of lumbar spinal cord segments (L4-L5) associated with injured nerve, but also in remote cervical segments (C6-C8). The increase in regeneration-associated proteins in the cervical DRG neurons was associated with the greater length of regenerated axons 1 day after ulnar nerve crush following prior sciatic nerve injury as compared to controls with only ulnar nerve crush. The increased axonal regeneration capacity of cervical DRG neurons after a prior conditioning sciatic nerve lesion was confirmed by neurite outgrowth assay of in vitro cultivated DRG neurons. Intrathecal injection of IL-6 or a JAK2 inhibitor (AG490) revealed a role for the IL-6 signaling pathway in activating the pro-regenerative state in remote DRG neurons. Our results suggest that the pro-regenerative state induced in the DRG neurons non-associated with the injured nerve reflects a systemic reaction of these neurons to unilateral sciatic nerve injury.

The Effect of Botulinum Neurotoxin Serotype a Heavy Chain on the Growth Related Proteins and Neurite Outgrowth after Spinal Cord Injury in Rats

(1) Background: The botulinum toxin A (BoNT-A) heavy chain (HC) can stimulate the growth of primary motor neurites. (2) Methods: A recombinant BoNT/A HC was injected locally plus interval intrathecal catheter of BoNT/A HC to rats with ipsilateral semi-dissociated lumbar spinal cord injuries (SCIs). First, 2D gel with a silver nitrate stain was applied to detect the general pattern of protein expression. Growth associated protein 43 (GAP-43) and superior cervical ganglion 10 (SCG10) were chosen to represent the altered proteins, based on their molecular weight and pI, and were used to further detect their expression. Meanwhile, the neuronal processes were measured. The measurements of thermal hyperalgesia and grasp power at the ipsilateral hindlimb were used to evaluate spinal sensory and motor function, respectively. (3) Results: The local injection of BoNT/A HC followed by its intrathecal catheter intervally altered the spinal protein expression pattern after an SCI; protein expression was similar to normal levels or displayed a remarkable increase. The changes in the expression and distribution of phosphorylated growth associated protein 43(p-GAP 43) and superior cervical ganglion 10 (SCG 10) indicated that the administration of BoNT/A HC to the SCI significantly amplified the expression of p-GAP43 and SCG10 (p < 0.05). Meanwhile, the positive immunofluorescent staining for both p-GAP43 and SCG10 was mainly present near the rostral aspect of the injury, both in the cytoplasm and the neuronal processes. Moreover, the outgrowth of neurites was stimulated by the BoNT/A HC treatment; this was evident from the increase in neurite length, number of branches and the percentage of cells with neuronal processes. The results from the spinal function tests suggested that the BoNT/A HC did not affect sensation, but had a large role in improving the ipsilateral hindlimb grasp power (p < 0.05). (4) Conclusions: The local injection with the intermittent intrathecal administration of BoNT/A heavy chain to rats with SCI increased the local expression of GAP-43 and SCG 10, which might be affiliated with the regeneration of neuronal processes surrounding the injury, and might also be favorable to the relief of spinal motor dysfunction.

Facilitating effects of Buyang Huanwu decoction on axonal regeneration after peripheral nerve transection

Ethnopharmacological relevanceIn traditional Asian medicine, Buyang Huanwu decoction (BYHWD) has been used for the treatment of cardiovascular and neurological disorders. Recent experimental studies have begun to provide evidence on the protective effects of BYHWD on injured peripheral nerves. Aim of the studyTo examine whether BYHWD was effective in inducing axonal regeneration after peripheral nerve transection, and if so, how it acted on the nerve. Materials and methodsThe sciatic nerve in rats was transected and resutured 0, 1, or 4 weeks later. BYHWD was orally administered daily into the animals with nerve transection and coaptation (NTC). Axonal regeneration was measured by immunofluorescence staining of NF-200 and superior cervical ganglion 10 (SCG10) and by retrograde tracing method. Changes of protein levels in the sciatic nerve were analyzed by western blot analysis. Effects of BYHWD and its constituents on neurite outgrowth were analyzed in cultured dorsal root ganglion (DRG) neurons. Hot plate and treadmill training tests were performed to assess the levels of functional recovery after nerve injury. ResultsThe rate of axonal regeneration was attenuated by delayed coaptation after transection, but improved by BYHWD treatment. Levels of phospho-Erk1/2 and Cdc2 phosphorylation of vimentin, measured as indicators of the activation of regenerating axons and supportive Schwann cells, were increased in the sciatic nerve of NTC animals, and their distribution in the proximal and distal nerves were affected by BYHWD treatment. Treatment of BYHWD during the period of chronic denervation significantly increased axonal regeneration when analyzed by immunofluorescence staining and retrograde tracing methods. Neurite outgrowth of DRG neurons cocultured with Schwann cells from the chronically transected sciatic nerves was enhanced by BYHWD treatment. Radix Paeoniae Rubra induced neurite outgrowth most efficiently among all herbal constituents of BYHWD. Finally, hot plate and treadmill training tests demonstrated that BYHWD administration significantly improved the sensorimotor nerve function in NTC animals. ConclusionsOur data suggest that BYHWD treatment may contribute to the timely interaction between regenerating axons and distal Schwann cells in the transected nerve and facilitate axonal regeneration.

WD40-repeat 47, a microtubule-associated protein, is essential for brain development and autophagy

The family of WD40-repeat (WDR) proteins is one of the largest in eukaryotes, but little is known about their function in brain development. Among 26 WDR genes assessed, we found 7 displaying a major impact in neuronal morphology when inactivated in mice. Remarkably, all seven genes showed corpus callosum defects, including thicker (Atg16l1, Coro1c, Dmxl2, and Herc1), thinner (Kif21b and Wdr89), or absent corpus callosum (Wdr47), revealing a common role for WDR genes in brain connectivity. We focused on the poorly studied WDR47 protein sharing structural homology with LIS1, which causes lissencephaly. In a dosage-dependent manner, mice lacking Wdr47 showed lethality, extensive fiber defects, microcephaly, thinner cortices, and sensory motor gating abnormalities. We showed that WDR47 shares functional characteristics with LIS1 and participates in key microtubule-mediated processes, including neural stem cell proliferation, radial migration, and growth cone dynamics. In absence of WDR47, the exhaustion of late cortical progenitors and the consequent decrease of neurogenesis together with the impaired survival of late-born neurons are likely yielding to the worsening of the microcephaly phenotype postnatally. Interestingly, the WDR47-specific C-terminal to LisH (CTLH) domain was associated with functions in autophagy described in mammals. Silencing WDR47 in hypothalamic GT1-7 neuronal cells and yeast models independently recapitulated these findings, showing conserved mechanisms. Finally, our data identified superior cervical ganglion-10 (SCG10) as an interacting partner of WDR47. Taken together, these results provide a starting point for studying the implications of WDR proteins in neuronal regulation of microtubules and autophagy.

Intervention with 7,8-dihydroxyflavone blocks further striatal terminal loss and restores motor deficits in a progressive mouse model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive neurological disorder and current therapies help alleviate symptoms, but are not disease modifying. In the flavonoid class of compounds, 7,8-dihydroxyflavone (7,8-DHF) has been reported to elicit tyrosine kinase receptor B (TrkB) dimerization and autophosphorylation that further stimulates signaling cascades to promote cell survival/growth, differentiation, and plasticity. In this study we investigated if 7,8-DHF could prevent further loss of dopaminergic cells and terminals if introduced at the midpoint (i.e. intervention) of our progressive mouse model of PD. In our model, 1-methyl-4phenyl-1,2,3,6-tetrahyrdopyridine (MPTP) is administered with increased doses each week (8, 16, 24, 32-kg/mg) over a 4-week period. We found that despite 4weeks of MPTP treatment, animals administered 7,8-DHF starting at the 2-week time period maintained 54% of the tyrosine hydroxylase (TH) levels within the dorsolateral (DL) striatum compared to the vehicle group, which was comparable to animals treated with MPTP for 2weeks and was significantly greater compared to animals treated with MPTP for the full 4weeks. Animals treated with MPTP and 7,8-DHF also demonstrated increased levels of, a sprouting-associated protein, superior cervical ganglion-10 (SCG10), phosphorylated TrkB (pTrkB), and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) within the DL striatum and substantia nigra (SN) compared to the 4-week MPTP-treated animals. In addition, motor deficits seen in the 2- and 4-week MPTP-treated animals were restored following administration of 7,8-DHF. We are reporting here for the first time that intervention with 7,8-DHF blocks further loss of dopaminergic terminals and restores motor deficits in our progressive MPTP mouse model. Our data suggest that 7,8-DHF has the potential to be a translational therapy in PD.

Miswiring the brain: Δ9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway.

Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly Δ(9)-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring.

Dynamic regulation of SCG10 in regenerating axons after injury

Peripheral axons can re-extend robustly after nerve injury. Soon after a nerve crush regenerating axons grow through the nerve segment distal to the lesion in close proximity to distal axons that are still morphologically and molecularly preserved. Hence, following the progress of regenerating axons necessitates markers that can distinguish between regenerating and degenerating axons. Here, we show that axonal levels of superior cervical ganglion 10 (SCG10) are dynamically regulated after axonal injury and provide an efficient method to label regenerating axons. In contrast to the rapid loss of SCG10 in distal axons (Shin et al., 2012b), we report that SCG10 accumulates in the proximal axons within an hour after injury, leading to a rapid identification of the lesion site. The increase in SCG10 levels is maintained during axon regeneration after nerve crush or nerve repair and allows for more selective labeling of regenerating axons than the commonly used markers growth-associated protein 43 (GAP43) and YFP. SCG10 is preferentially expressed in regenerating sensory axons rather than motor axons in the sciatic nerve. In a mouse model of slow Wallerian degeneration, SCG10 labeling remains selective for regenerating axons and allows for a quantitative analysis of delayed regeneration in this mutant. Taken together, these data demonstrate the utility of SCG10 as an efficient and selective marker of sensory axon regeneration.

SCG10 is a JNK target in the axonal degeneration pathway.

Axons actively self-destruct following genetic, mechanical, metabolic, and toxic insults, but the mechanism of axonal degeneration is poorly understood. The JNK pathway promotes axonal degeneration shortly after axonal injury, hours before irreversible axon fragmentation ensues. Inhibition of JNK activity during this period delays axonal degeneration, but critical JNK substrates that facilitate axon degeneration are unknown. Here we show that superior cervical ganglion 10 (SCG10), an axonal JNK substrate, is lost rapidly from mouse dorsal root ganglion axons following axotomy. SCG10 loss precedes axon fragmentation and occurs selectively in the axon segments distal to transection that are destined to degenerate. Rapid SCG10 loss after injury requires JNK activity. The JNK phosphorylation sites on SCG10 are required for its rapid degradation, suggesting that direct JNK phosphorylation targets SCG10 for degradation. We present a mechanism for the selective loss of SCG10 distal to the injury site. In healthy axons, SCG10 undergoes rapid JNK-dependent degradation and is replenished by fast axonal transport. Injury blocks axonal transport and the delivery of SCG10, leading to the selective loss of the labile SCG10 distal to the injury site. SCG10 loss is functionally important: Knocking down SCG10 accelerates axon fragmentation, whereas experimentally maintaining SCG10 after injury promotes mitochondrial movement and delays axonal degeneration. Taken together, these data support the model that SCG10 is an axonal-maintenance factor whose loss is permissive for execution of the injury-induced axonal degeneration program.

Reduced Myelin Basic Protein and Actin-Related Gene Expression in Visual Cortex in Schizophrenia

Most brain gene expression studies of schizophrenia have been conducted in the frontal cortex or hippocampus. The extent to which alterations occur in other cortical regions is not well established. We investigated primary visual cortex (Brodmann area 17) from the Stanley Neuropathology Consortium collection of tissue from 60 subjects with schizophrenia, bipolar disorder, major depression, or controls. We first carried out a preliminary array screen of pooled RNA, and then used RT-PCR to quantify five mRNAs which the array identified as differentially expressed in schizophrenia (myelin basic protein [MBP], myelin-oligodendrocyte glycoprotein [MOG], β-actin [ACTB], thymosin β-10 [TB10], and superior cervical ganglion-10 [SCG10]). Reduced mRNA levels were confirmed by RT-PCR for MBP, ACTB and TB10. The MBP reduction was limited to transcripts containing exon 2. ACTB and TB10 mRNAs were also decreased in bipolar disorder. None of the transcripts were altered in subjects with major depression. Reduced MBP mRNA in schizophrenia replicates findings in other brain regions and is consistent with oligodendrocyte involvement in the disorder. The decreases in expression of ACTB, and the actin-binding protein gene TB10, suggest changes in cytoskeletal organisation. The findings confirm that the primary visual cortex shows molecular alterations in schizophrenia and extend the evidence for a widespread, rather than focal, cortical pathophysiology.

IKAP/Elp1 involvement in cytoskeleton regulation and implication for familial dysautonomia

Deficiency in the IKAP/Elp1 protein leads to the recessive sensory autosomal congenital neuropathy which is called familial dysautonomia (FD). This protein was originally identified as a role player in transcriptional elongation being a subunit of the RNAPII transcriptional Elongator multi-protein complex. Subsequently, IKAP/Elp1 was shown to play various functions in the cytoplasm. Here, we describe experiments performed with IKAP/Elp1 downregulated cell lines and FD-derived cells and tissues. Immunostaining of the cytoskeleton component α-tubulin in IKAP/Elp1 downregulated cells revealed disorganization of the microtubules (MTs) that was reflected in aberrant cell shape and process formation. In contrast to a recent report on the decrease in α-tubulin acetylation in IKAP/Elp1 downregulated cells, we were unable to observe any effect of IKAP/Elp1 deficiency on α-tubulin acetylation in the FD cerebrum and in a variety of IKAP/Elp1 downregulated cell lines. To explore possible candidates involved in the observed aberrations in MTs, we focused on superior cervical ganglion-10 protein (SCG10), also called STMN2, which is known to be an MT destabilizing protein. We have found that SCG10 is upregulated in the IKAP/Elp1-deficient FD cerebrum, FD fibroblasts and in IKAP/Elp1 downregulated neuroblastoma cell line. To better understand the effect of IKAP/Elp1 deficiency on SCG10 expression, we investigated the possible involvement of RE-1-silencing transcription factor (REST), a known repressor of the SCG10 gene. Indeed, REST was downregulated in the IKAP/Elp1-deficient FD cerebrum and IKAP/Elp1 downregulated neuroblastoma cell line. These results could shed light on a possible link between IKAP/Elp1 deficiency and cytoskeleton destabilization.

A Direct Redox Regulation of Protein Kinase C Isoenzymes Mediates Oxidant-induced Neuritogenesis in PC12 Cells

In this study, we have used the PC12 cell model to elucidate the mechanisms by which sublethal doses of oxidants induce neuritogenesis. The xanthine/xanthine oxidase (X/XO) system was used for the steady state generation of superoxide, and CoCl(2) was used as a representative transition metal redox catalyst. Upon treatment of purified protein kinase C (PKC) with these oxidants, there was an increase in its cofactor-independent activation. Redox-active cobalt competed with the redoxinert zinc present in the zinc-thiolates of the PKC regulatory domain and induced the oxidation of these cysteine-rich regions. Both CoCl(2) and X/XO induced neurite outgrowth in PC12 cells, as determined by an overexpression of neuronal marker genes. Furthermore, these oxidants induced a translocation of PKC from cytosol to membrane and subsequent conversion of PKC to a cofactor-independent form. Isoenzyme-specific PKC inhibitors demonstrated that PKCepsilon plays a crucial role in neuritogenesis. Moreover, oxidant-induced neurite outgrowth was increased with a conditional overexpression of PKCepsilon and decreased with its knock-out by small interfering RNA. Parallel with PKC activation, an increase in phosphorylation of the growth-associated neuronal protein GAP-43 at Ser(41) was observed. Additionally, there was a sustained activation of extracellular signal-regulated kinases 1 and 2, which was correlated with activating phosphorylation (Ser(133)) of cAMP-responsive element-binding protein. All of these signaling events that are causally linked to neuritogenesis were blocked by antioxidant N-acetylcysteine (both L and D-forms) and by a variety of PKC-specific inhibitors. Taken together, these results strongly suggest that sublethal doses of oxidants induce neuritogenesis via a direct redox activation of PKCepsilon.

Superior cervical ganglion-10 protein as a molecular effector of c-Jun N-terminal kinase 1: implications for the therapeutic targeting of Jun N-terminal kinase in nerve regeneration

Background: Cell stress and tissue injury lead to c-Jun N-terminal kinase (JNK) activation, which is known to contribute to cell death. Paradoxically, strong evidence supports an important role for JNK in the regeneration of neuronal processes, subsequent to injury. Objective: Recent research revealed the growth cone-associated protein superior cervical ganglion-10 protein as a candidate effector for the regeneration pathway mediated by JNK1. This implies that neuroprotective strategies targeting JNK may have negative effects on neuronal regeneration, unless JNK1 function is spared, and that the mechanistic relationships between JNK1 and neuronal regeneration deserve increased attention. Results: This review proposes a model reconciling the microtubule regulatory properties of superior cervical ganglion protein 10 with its role as a JNK effector of regeneration and highlight remaining issues to be resolved.

SCLIP, a Microtubule-destabilizing Factor, Interacts with RasGRF1 and Inhibits Its Ability to Promote Rac Activation and Neurite Outgrowth

RasGRF1 is a neuron-specific guanine nucleotide exchange factor for the small GTPases Ras and Rac. It is implicated in the regulation of memory formation and in the development of tolerance to drug abuse, although the mechanisms have been elucidated only in part. Here we report the isolation, by the yeast two-hybrid screen, of the microtubule-destabilizing factor SCLIP (SCG10-like protein) as a novel RasGRF1-interacting protein. This interaction requires the region spanning the Dbl-homology domain of RasGRF1, endowed with catalytic activity on Rac. In search for a possible function we found by biochemical means that SCLIP influences the signaling properties of RasGRF1, greatly reducing its ability to activate the Rac/p38 MAPK pathway, while the Ras/Erk one remains unaffected. Moreover, a potential role is suggested by transfection studies in neuronal PC12 cells in which RasGRF1 induces neurite outgrowth, and coexpression of SCLIP counteracts this effect, causing a dramatic decrease in the percentage of cells bearing neurites, which also appear significantly shortened. This study unveils a physical and functional interaction between RasGRF1 and SCLIP. We suggest that this novel interplay may have possible implications in mechanisms that regulate neuronal morphology and structural plasticity.

A newly modified SCG10 promoter and Cre/loxP-mediated gene amplification system achieve highly specific neuronal expression in animal brains

We designed a new promoter that drives transgene expression in an exclusively neuron-specific manner. The promoter of superior cervical ganglion10 (SCG10), expressed in neurons, was further modified to enhance its neuron specificity and activity by changing its length and fusing a multiple neuronal restrictive silencer element (NRSE) to its upstream or downstream regions. The promoter, which contained 2 kb original promoter length and two extra NRSEs in its downstream region, eventually exhibited remarkable neuron specificity as well as strong activity. To further amplify the promoter activity, the promoter was introduced into a Cre recombinase (Cre)-expressing adenovirus, and subsequent combination with Cre-inducible enhanced green fluorescence protein (EGFP)-expressing adenovirus vector, which has much stronger general promoter, resulted in a remarkably strong gene expression exclusively in neuronal cells of mixed cultures and in an animal model. This system is also applicable to astrocyte-specific expression; for instance, by changing the Cre promoter cassette to an astrocyte-specific promoter. The present relatively compact promoter combined with Cre/loxP system could be useful for a wide range of transgene experiments in vivo as well as for clinical applications.

Differential regulation of the growth-associated proteins GAP-43 and superior cervical ganglion 10 in response to lesions of the cortex and substantia nigra in the adult rat

Investigation of the elements underlying synapse replacement after brain injury is essential for predicting the neural compensation that can be achieved after various types of damage. The growth-associated proteins superior cervical ganglion-10 and growth-associated protein-43 have previously been linked with structural changes in the corticostriatal system in response to unilateral deafferentation. To examine the regulation of this response, unilateral cortical aspiration lesion was carried out in combination with ipsilateral 6-hydroxydopamine lesion of the substantia nigra, and the time course of the contralateral cortical molecular response was followed. Unilateral cortical aspiration lesion in rats corresponds with an upregulation of superior cervical ganglion-10 mRNA at 3 and 10 days post-lesion, and protein, sustained from three to at least 27 days following lesion. With the addition of substantia nigra lesion, the response shifts to an upregulation of growth-associated protein-43 mRNA at 3 and 10 days post-lesion, and protein after 10 days. Nigral lesion alone does not alter contralateral expression of either gene. Likewise, motor function assessment using the rotorod test revealed no significant long-term deficits in animals that sustained only nigrostriatal damage, but cortical lesion was associated with a temporary deficit which was sustained when nigrostriatal input was also removed. Growth-associated protein-43 and superior cervical ganglion-10, two presynaptic genes that are postulated to play roles in lesion-induced sprouting, are differentially upregulated in corticostriatal neurons after cortical versus combined cortical/nigral lesions. The shift in contralateral gene response from superior cervical ganglion-10 to growth-associated protein-43 upregulation and associated behavioral deficit following combined cortical and nigral denervation suggest that nigrostriatal afferents regulate cortical lesion-induced gene expression and ultimate functional outcome.

Lesion-induced sprouting of commissural/associational axons and induction of GAP-43 mRNA in hilar and CA3 pyramidal neurons in the hippocampus are diminished in aged rats

Removal of synaptic connections following a partial deafferentation lesion results in a sprouting of remaining afferents that terminate near the denervated area. However, while the ability to form new synapses in response to injury has been reported in both young and aged rats, previous studies have suggested that the injury-induced response in the hippocampus of aged rats may be delayed and/or not as extensive as compared to young adults. Given that growth associated proteins are central for the regulation of neurite outgrowth during both development and regeneration, we were interested in determining if the magnitude and time course of the sprouting response of hippocampal neurons to deafferentation might correlate with induction of growth associated proteins and whether these parameters could be modulated with age. For our studies we used the Holmes fiber stain to determine the expansion of the C/A fiber plexus following denervation and compared the time course of the sprouting response with that observed by in situ hybridization for the neurite outgrowth proteins, growth associated protein-43 (GAP-43), superior cervical ganglion-10 (SCG-10), and neurofilament-68 (NF-68) at various time points after the lesion for each age group. We found that the commissural/associational (C/A) fiber plexus expanded by 45% in young adult rats at 30 and 45 d postlesion and was accompanied by a significant increase in expression of GAP-43 mRNA in both ipsilateral and contralateral hilar and CA3 pyramidal neurons, the cell bodies of origin for the C/A pathway. In contrast, a dampened sprouting response was observed in aged rats at all time points postlesion and coincided with a lack of induction of any of the growth-associated proteins. These results suggest that GAP-43 is involved in outgrowth of C/A axons in the hippocampus in response to a partial deafferentation lesion. However, factors that stimulate neurite outgrowth and upregulate GAP-43 mRNA in response to a partial deafferentation lesion diminish with age.