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a–h , Response of Purkinje cells after intracerebellar injections of colchicine. Micrograph a shows the morphological alterations of anti-calbindin-immunolabeled Purkinje cells, which mostly involve the formation of axonal torpedoes ( arrowheads ). Starting from the very first days after injection, numerous Purkinje cells ( arrowheads in b–d ) show a strong nuclear staining for c-Jun ( b ), P-Jun ( d ), and JunD ( c ) immediate early genes. In addition, c-Jun expression is increased in granule cells ( b ). A survey picture of a cerebellar section labeled for NADPH diaphorase histochemistry 14 d after colchicine injection is shown in e . The arrow points to the area of cortical atrophy around the injection site. Note, however, ( Figure legend continues )
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Axon regeneration requires that injured neurons reinitiate long-distance growth and upregulate specific genes. To address the question of whether inhibitory environmental cues along the axon could exert a negative, tonic downregulation of growth-associated genes, we have examined adult rat Purkinje cells, which are endowed with poor regenerative ca...
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... test this hypothesis, we blocked axonal transport with col- chicine. Colchicine injection into the intact cerebellum in vivo induced a strong expression of c-Jun (Fig. 4b), P-Jun (Fig. 4d), and JunD (Fig. 4c) in numerous Purkinje cells distributed throughout several lobules around the injection site (Fig. 5). Quantitative estimation of c-Jun expression showed that several hundreds of reactive Purkinje cells per section were present from the very first days after lesion (Fig. 5). This expression was ...
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... test this hypothesis, we blocked axonal transport with col- chicine. Colchicine injection into the intact cerebellum in vivo induced a strong expression of c-Jun (Fig. 4b), P-Jun (Fig. 4d), and JunD (Fig. 4c) in numerous Purkinje cells distributed throughout several lobules around the injection site (Fig. 5). Quantitative estimation of c-Jun expression showed that several hundreds of reactive Purkinje cells per section were present from the very first days after lesion (Fig. 5). This expression was maintained for several ...
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... test this hypothesis, we blocked axonal transport with col- chicine. Colchicine injection into the intact cerebellum in vivo induced a strong expression of c-Jun (Fig. 4b), P-Jun (Fig. 4d), and JunD (Fig. 4c) in numerous Purkinje cells distributed throughout several lobules around the injection site (Fig. 5). Quantitative estimation of c-Jun expression showed that several hundreds of reactive Purkinje cells per section were present from the very first days after lesion (Fig. 5). This expression was maintained for several weeks, and values ...
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... estimation of c-Jun expression showed that several hundreds of reactive Purkinje cells per section were present from the very first days after lesion (Fig. 5). This expression was maintained for several weeks, and values returned to control levels after 2 months. Colchicine application also induced a strong NADPH diaphorase reactivity (see Fig. 4e,f ) and CAP-23 immunolabeling (see Fig. 4g,h) in the same cerebellar lobuli. In contrast, Purkinje cells did not show GAP-43 immunostaining or GAP-43 mRNA upregulation (data not shown). The appearance of CAP-23 expression and NADPH reactivity was delayed with respect to the immediate early genes but was well evident at 7 and 14 d (Fig. ...
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... that several hundreds of reactive Purkinje cells per section were present from the very first days after lesion (Fig. 5). This expression was maintained for several weeks, and values returned to control levels after 2 months. Colchicine application also induced a strong NADPH diaphorase reactivity (see Fig. 4e,f ) and CAP-23 immunolabeling (see Fig. 4g,h) in the same cerebellar lobuli. In contrast, Purkinje cells did not show GAP-43 immunostaining or GAP-43 mRNA upregulation (data not shown). The appearance of CAP-23 expression and NADPH reactivity was delayed with respect to the immediate early genes but was well evident at 7 and 14 d (Fig. 5). At 2 months after injection, NADPH ...
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... CAP-23 expression and NADPH reactivity was delayed with respect to the immediate early genes but was well evident at 7 and 14 d (Fig. 5). At 2 months after injection, NADPH diaphorase- reactive cells were absent from three of the four examined ani- mals, although one of them still displayed sparse labeled cells. CAP-23-immunoreactive neurons (see Fig. 4g,h) were consis- tently present between 1 and 2 weeks after injury, although their number was always far fewer than that of neurons labeled for the other markers. As a control experiment we injected -lumicolchicine, which did not induce any expression of the examined markers in Purkinje cells except for a few neurons located around the ...
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... previously reported (Pioro and Cuello, 1988), colchicine treatment induced structural abnormalities in Purkinje cell axons, mostly involving the appearance of torpedoes (see Fig. 4a). In addition, because of its well established neurotoxic effect (Goldschmidt and Steward, 1982) an area of cortical atrophy and neuronal degeneration was evident around the injection site (Fig. 4e). However, this area was much smaller than the area in which marker expression was induced. Indeed, the distribution of reac- tive Purkinje ...
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... and Cuello, 1988), colchicine treatment induced structural abnormalities in Purkinje cell axons, mostly involving the appearance of torpedoes (see Fig. 4a). In addition, because of its well established neurotoxic effect (Goldschmidt and Steward, 1982) an area of cortical atrophy and neuronal degeneration was evident around the injection site (Fig. 4e). However, this area was much smaller than the area in which marker expression was induced. Indeed, the distribution of reac- tive Purkinje cells in the treated cerebella was consistent with a diffusion of colchicine along the axial white matter of several ...
Citations
... Immunodetection after in situ hybridization on adult (18 months old) female brain sections was performed using 3,3Ј diaminobenzidine (DAB)chromogen (Sigma-Aldrich) to visualize signals as brown precipitates ( Fig. 1C) according to the procedure as previously described (Matsui et al., 2014). For the immunostaining of mouse organotypic cerebellar slices 6 d after transfection [8 d in vitro (DIV)], the slices were treated as described previously (Zagrebelsky et al., 1998). ...
... Preparation of cerebellar organotypic cultures and particle-mediated gene transfer. Cerebellar organotypic cultures (thickness of 400 m) were prepared from postnatal day (P)10 C57BL/ 6 mice of either sex as previously described (Stoppini et al., 1991;Zagrebelsky et al., 1998). Individual neurons were biolistically transfected after 2 DIV using the Helios Gene Gun System (Bio-Rad) with the cpce-CMV mini :GFP and CMV enhancer/promoter (CMV):mCherry constructs. ...
Purkinje cells (PCs) are primarily affected in neurodegenerative spinocerebellar ataxias (SCAs). For generating animal models for SCAs, genetic regulatory elements specifically targeting PCs are required, thereby linking pathological molecular effects with impaired function and organismic behavior. Because cerebellar anatomy and function are evolutionary conserved, zebrafish represent an excellent model to study SCAs in vivo. We have isolated a 258 bp cross-species PC-specific enhancer element that can be used in a bidirectional manner for bioimaging of transgene-expressing PCs in zebrafish (both sexes) with variable copy numbers for tuning expression strength. Emerging ectopic expression at high copy numbers can be further eliminated by repurposing microRNA-mediated posttranslational mRNA regulation.
Subsequently, we generated a transgenic SCA type 13 (SCA13) model, using a zebrafish-variant mimicking a human pathological SCA13R420H mutation, resulting in cell-autonomous progressive PC degeneration linked to cerebellum-driven eye-movement deficits as observed in SCA patients. This underscores that investigating PC-specific cerebellar neuropathologies in zebrafish allows for interconnecting bioimaging of disease mechanisms with behavioral analysis suitable for therapeutic compound testing.
... In addition, those mice showed severe ataxic symptoms, indicating that climbing fiber input on Purkinje cell activity is fundamental to maintain motor control [17]. Thereafter, De Zeeuw remembered other important contributions that Ferdinando gave for the understanding of Purkinje cell plasticity [18][19][20] and mentioned Ferdinando's study showing that enriched environment triggers remarkable remodelling of Purkinje cell axon terminals and precerebellar afferents in the cerebellar nuclei [21]. This work offered the fundamental frame for the recent pavlovian eyeblink conditioning experiments conducted by De Zeeuw, who found substantial axonal growth in the cerebellar nuclei of adult mice during that form of motor learning [22]. ...
To remember our friend and colleague Ferdinando Rossi, prematurely passed away on 24th January 2014, a symposium was held during the ninth FENS meeting in Milan. It was focused on the development and plasticity of the cerebellum, the main topics of Ferdinando's research. From the talks of the invited speakers, Giacomo Consalez, Karl Schilling, Alain Chédotal, and Chris De Zeeuw, it clearly emerged that Ferdinando had a huge impact on the research of many scientists like them, as well as in the whole field of brain development and regeneration. With this symposium, we celebrated a brilliant scientist, devoted to Neuroscience with tireless passion and curiosity.
... Anti-Nogo-A antibodies promote neuroplasticity by overcoming inhibitory signals from oligodendocytes and reducing growth cone collapse, thereby limiting a major source of growth inhibition in the damaged CNS and promoting a suitable environment for beneficial plasticity. Inhibiting Nogo-A has been shown to enhance expression of growth-promoting factors, such as GAP-43, and induce axonal sprouting of CST fibres across the midline of the cervical spinal cord Zagrebelsky et al., 1998). Treatment was effective even when animals exhibited common comorbidities generally associated with poor stroke recovery, such as hypertension (Wiessner et al., 2003). ...
... Recent studies demonstrate that extrinsic cues from myelin-associated molecules, such as Nogo A, exert a constitutive retrograde inhibitory signal that dampens both this cell body response and the spontaneous inclination for axonal sprouting in Purkinje cells ( Rossi, 2004, 2005). Blocking Nogo A with neutralizing antibodies in cerebellum induces both injury/growth associated molecules and profuse sprouting along the intracortical segment of Purkinje cell axons in the recurrent collateral plexus (Zagrebelsky et al., 1998; Foscarin et al., 2009). Long-term injured Purkinje cell axons tend to lose myelin along their intracortical course, where some sprouting occurs (Gianola and Rossi, 2002). ...
Growing clinical, neuro-imaging and post-mortem data have implicated the cerebellum as playing an important role in the pathogenesis of essential tremor. Aside from a modest reduction of Purkinje cells in some post-mortem studies, Purkinje cell axonal swellings (torpedoes) are present to a greater degree in essential tremor cases than controls. Yet a detailed study of more subtle morphometric changes in the Purkinje cell axonal compartment has not been undertaken. We performed a detailed morphological analysis of the Purkinje cell axonal compartment in 49 essential tremor and 39 control brains, using calbindin D28k immunohistochemistry on 100-µm cerebellar cortical vibratome tissue sections. Changes in axonal shape [thickened axonal profiles (P = 0.006), torpedoes (P = 0.038)] and changes in axonal connectivity [axonal recurrent collaterals (P < 0.001), axonal branching (P < 0.001), terminal axonal sprouting (P < 0.001)] were all present to an increased degree in essential tremor cases versus controls. The changes in shape and connectivity were significantly correlated [e.g. correlation between thickened axonal profiles and recurrent collaterals (r = 0.405, P < 0.001)] and were correlated with tremor duration among essential tremor cases with age of onset >40 years. In essential tremor cases, thickened axonal profiles, axonal recurrent collaterals and branched axons were 3- to 5-fold more frequently seen on the axons of Purkinje cells with torpedoes versus Purkinje cells without torpedoes. We document a range of changes in the Purkinje cell axonal compartment in essential tremor. Several of these are likely to be compensatory changes in response to Purkinje cell injury, thus illustrating an important feature of Purkinje cells, which is that they are relatively resistant to damage and capable of mobilizing a broad range of axonal responses to injury. The extent to which this plasticity of the Purkinje cell axon is partially neuroprotective or ultimately ineffective at slowing further cellular changes and cell death deserves further study in essential tremor.
... At the molecular level, these structural changes are accompanied by a concomitant upregulation of growthassociated markers and transcription factors, which suggests that Nogo-A may actively suppress anatomical plasticity in the adult CNS by a tonic downregulation of growth-associated gene expression (10, 14, 22, 140) (FIGURE 2). Indeed, transcriptional profiling of hippocampal or cerebellar slices treated with Nogo-A-neutralizing antibodies as well as proteomic profiling of the CNS of adult Nogo-A KO mice pointed to a marked regulation of the growth cone cytoskeleton machinery and of growth-associated transcription factors toward increased growth (22, 82,140). Tonic growth inhibition might result from retrogradely transported inhibitory signals from the axons to the cell bodies. ...
Nogo-A was initially discovered as a myelin-associated growth inhibitory protein limiting axonal regeneration after central nervous system (CNS) injury. This review summarizes current knowledge on how myelin and neuronal Nogo-A and its receptors exert physiological functions ranging from the regulation of growth suppression to synaptic plasticity in the developing and adult intact CNS.
... Neurons differ widely in regard to their response to axonal injuries (Carulli et al., 2004;Dusart et al., 2005). For example, in the cerebellum, PCs respond to injury with little upregulation of plasticity-related genes in the cell body, no axonal regeneration after axotomy, and weak sprouting; most PCs survive, but they usually do not increase the expression of plasticity-related genes, except when the axotomy occurs near the cell body Bravin et al., 1997;Zagrebelsky et al., 1998;Wehrle et al., 2001;Morel et al., 2002;Gianola and Rossi, 2004). Further, axonal sprouting is limited and might be induced only following proper manipulation of intrinsic and environmental factors (Buffo et al., 1997(Buffo et al., , 2000Zagrebelsky et al., 1998;Zhang et al., 2005Zhang et al., , 2007. ...
... For example, in the cerebellum, PCs respond to injury with little upregulation of plasticity-related genes in the cell body, no axonal regeneration after axotomy, and weak sprouting; most PCs survive, but they usually do not increase the expression of plasticity-related genes, except when the axotomy occurs near the cell body Bravin et al., 1997;Zagrebelsky et al., 1998;Wehrle et al., 2001;Morel et al., 2002;Gianola and Rossi, 2004). Further, axonal sprouting is limited and might be induced only following proper manipulation of intrinsic and environmental factors (Buffo et al., 1997(Buffo et al., , 2000Zagrebelsky et al., 1998;Zhang et al., 2005Zhang et al., , 2007. ...
Structural plasticity occurs physiologically or after brain damage to adapt or re-establish proper synaptic connections. This capacity depends on several intrinsic and extrinsic determinants that differ between neuron types. We reviewed the significant endogenous regenerative potential of the neurons of the inferior olive (IO) in the adult rodent brain and the structural remodeling of the terminal arbor of their axons, the climbing fiber (CF), under various experimental conditions, focusing on the growth-associated protein GAP-43. CFs undergo remarkable collateral sprouting in the presence of denervated Purkinje cells (PCs) that are available for new innervation. In addition, severed olivo-cerebellar axons regenerate across the white matter through a graft of embryonic Schwann cells. In contrast, CFs undergo a regressive modification when their target is deleted. In vivo knockdown of GAP-43 in olivary neurons, leads to the atrophy of their CFs and a reduction in the ability to sprout toward surrounding denervated PCs. These findings demonstrate that GAP-43 is essential for promoting denervation-induced sprouting and maintaining normal CF architecture.
... Functional recovery in stroke is accompanied by plasticity of cortical efferents from the unlesioned hemisphere to the striatum, red nucleus, pons and cervical spinal cord. In vivo and in vitro studies have shown enhanced expression of growth promoting genes including c-Jun and GAP-43 in presence of IN-1 [68,69] and these probably occur in sprouting neurons in response to released inhibition of growth. ...
Neurorestorative therapies for stroke aim to reverse disability by reparative mechanisms (rather than to thrombolyse or to neuroprotect). A substantial and persuasive body of pre-clinical evidence has come from the evaluation of antibodies against Nogo-A (a myelin-associated inhibitor of plasticity) in rat models of stroke. Particularly impressive is the benefit of this therapy in models of permanent middle cerebral artery occlusion (MCAO) when given to elderly animals after a one week delay, in adult rats with co-morbidities, and in adult rats when treatment is delayed by up to 9 weeks after stroke (although antibodies against Nogo-A did not reverse disability in mice after proximal MCAO with reperfusion). We predict that antibodies against Nogo-A will improve outcome when combined with suitable additional rehabilitation, and also that antibodies against Nogo-A will improve outcome in animal models of haemmorhagic stroke that affect the same brain regions as ischemic stroke caused by MCAO. Antibodies against Nogo-A have been shown to be safe in Phase I clinical trials for acute spinal cord injury, and this may eventually facilitate a trial in stroke.
... How does axon injury lead to c-Jun expression and/or activation? Treatment with antibodies against the neurite growth inhibitor Nogo increase c-Jun expression in Purkinje cells, as does application of colchicine to block axonal transport, suggesting an inhibitory signal may be retrogradely transported from the axon to stop c-Jun expression and regenerationassociated genes (Zagrebelsky et al., 1998). On the other hand, c-Jun N-terminal kinase (JNK) is retrogradely transported in injured axons to activate c-Jun in the nucleus after peripheral nerve injury, as blockade of retrograde transport inhibits c-Jun activation (Lindwall and Kanje, 2005). ...
Understanding axon regenerative failure remains a major goal in neuroscience, and reversing this failure remains a major goal for clinical neurology. Although an inhibitory central nervous system environment clearly plays a role, focus on molecular pathways within neurons has begun to yield fruitful insights. Initial steps forward investigated the receptors and signaling pathways immediately downstream of environmental cues, but recent work has also shed light on transcriptional control mechanisms that regulate intrinsic axon growth ability, presumably through whole cassettes of gene target regulation. Here we will discuss transcription factors that regulate neurite growth in vitro and in vivo, including p53, SnoN, E47, cAMP-responsive element binding protein (CREB), signal transducer and activator of transcription 3 (STAT3), nuclear factor of activated T cell (NFAT), c-Jun activating transcription factor 3 (ATF3), sex determining region Ybox containing gene 11 (Sox11), nuclear factor κ-light chain enhancer of activated B cells (NFκB), and Krüppel-like factors (KLFs). Revealing the similarities and differences among the functions of these transcription factors may further our understanding of the mechanisms of transcriptional regulation in axon growth and regeneration.
... Anti-Nogo-A treatment of the adult CNS also enhanced growth and plasticity in the absence of an injury. Intact purkinje cells of the cerebellum reacted to a single injection of anti-Nogo-A antibody by up-regulating the expression of transcription factors and immediate early genes important for growth ( Zagrebelsky et al., 1998) and by profuse axonal sprouting ( Buffo et al., 2000). In addition, anti-Nogo-A treatment of intact, naive rats enhanced sprouting of CST fibres without affecting the gross behaviour of the animals ( Bareyre et al., 2002). ...
Following spinal cord injury (SCI) the adult central nervous system (CNS) has a limited but substantial capacity for repair and plastic reorganisation. The degree of reorganisation is determined by a number of factors such as the extent and location of the lesion, the remaining circuit activity within the CNS and the age at injury. However, even in the best cases this spontaneous reorganisation does not lead to full recovery of the affected behaviour but instead often results in a functionally successful but compensatory strategy. Current SCI research focuses on enhancing fibre tract (re-)growth and recovery processes. Two currently promising approaches are the neutralisation of CNS growth inhibitory factors, and rehabilitative training of remaining networks. Independently, both approaches can lead to substantial functional recovery and anatomical reorganisation. In this review we focus on Nogo-A, a neurite growth inhibitory protein present in the adult CNS, and its role in regenerative and plastic growth following SCI. We then discuss the efforts of rehabilitative training and the potential combination of the two therapies.
... The distance of the lesion site from the cell body is one of the factors determining neuronal responses to injury. For some populations of neurons, a more proximal axotomy leads to greater regenerative response by the cell body (567 and references cited therein). Lesion distance was also shown to influence specific molecular responses to injury, including activation of cell body kinases [8] and up-regulation of growth-associated genes [5,9101112. ...
... In specific neuron types, once distance between cell body and site of injury drops below a certain lower threshold, no regeneration occurs, whereas above this threshold the probability of regeneration increases continuously with the increase in distance between the cell body and site of injury [14,15]. In other neuronal populations, a more proximal axotomy leads to greater regenerative response by the cell body567. Despite the clear biological significance of injury distance in neural tissues, the mechanism by which distance fromFigure 7. Comparing two dynein velocity data sets. ...
Injury to nerve axons induces diverse responses in neuronal cell bodies, some of which are influenced by the distance from the site of injury. This suggests that neurons have the capacity to estimate the distance of the injury site from their cell body. Recent work has shown that the molecular motor dynein transports importin-mediated retrograde signaling complexes from axonal lesion sites to cell bodies, raising the question whether dynein-based mechanisms enable axonal distance estimations in injured neurons? We used computer simulations to examine mechanisms that may provide nerve cells with dynein-dependent distance assessment capabilities. A multiple-signals model was postulated based on the time delay between the arrival of two or more signals produced at the site of injury-a rapid signal carried by action potentials or similar mechanisms and slower signals carried by dynein. The time delay between the arrivals of these two types of signals should reflect the distance traversed, and simulations of this model show that it can indeed provide a basis for distance measurements in the context of nerve injuries. The analyses indicate that the suggested mechanism can allow nerve cells to discriminate between distances differing by 10% or more of their total axon length, and suggest that dynein-based retrograde signaling in neurons can be utilized for this purpose over different scales of nerves and organisms. Moreover, such a mechanism might also function in synapse to nucleus signaling in uninjured neurons. This could potentially allow a neuron to dynamically sense the relative lengths of its processes on an ongoing basis, enabling appropriate metabolic output from cell body to processes.
