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1.
Nature ; 574(7779): 559-564, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645735

RESUMO

Although glucose-sensing neurons were identified more than 50 years ago, the physiological role of glucose sensing in metazoans remains unclear. Here we identify a pair of glucose-sensing neurons with bifurcated axons in the brain of Drosophila. One axon branch projects to insulin-producing cells to trigger the release of Drosophila insulin-like peptide 2 (dilp2) and the other extends to adipokinetic hormone (AKH)-producing cells to inhibit secretion of AKH, the fly analogue of glucagon. These axonal branches undergo synaptic remodelling in response to changes in their internal energy status. Silencing of these glucose-sensing neurons largely disabled the response of insulin-producing cells to glucose and dilp2 secretion, disinhibited AKH secretion in corpora cardiaca and caused hyperglycaemia, a hallmark feature of diabetes mellitus. We propose that these glucose-sensing neurons maintain glucose homeostasis by promoting the secretion of dilp2 and suppressing the release of AKH when haemolymph glucose levels are high.


Assuntos
Encéfalo/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Glucagon/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Neurônios/metabolismo , Animais , Axônios/metabolismo , Encéfalo/anatomia & histologia , Drosophila melanogaster/anatomia & histologia , Glucose/análise , Hormônios de Inseto/metabolismo , Masculino , Inibição Neural , Vias Neurais , Neuropeptídeos/química , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Oligopeptídeos/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Ácido Pirrolidonocarboxílico/metabolismo
2.
Nat Cell Biol ; 21(9): 1086-1092, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481789

RESUMO

Tau is an intrinsically disordered protein, which diffuses on microtubules1. In neurodegenerative diseases, collectively termed tauopathies, malfunction of tau and its detachment from axonal microtubules are correlated with axonal degeneration2. Tau can protect microtubules from microtubule-degrading enzymes such as katanin3. However, how tau carries out this regulatory function is still unclear. Here, using in vitro reconstitution, we show that tau molecules on microtubules cooperatively form cohesive islands that are kinetically distinct from tau molecules that individually diffuse on microtubules. Dependent on the tau concentration in solution, the islands reversibly grow or shrink by addition or release of tau molecules at their boundaries. Shielding microtubules from kinesin-1 motors and katanin, the islands exhibit regulatory qualities distinct from a comparably dense layer of diffusible tau. Superprocessive kinesin-8 motors penetrate the islands and cause their disassembly. Our results reveal a microtubule-dependent phase of tau that constitutes an adaptable protective layer on the microtubule surface. We anticipate that other intrinsically disordered axonal proteins display a similar cooperative behaviour and potentially compete with tau in regulating access to the microtubule surface.


Assuntos
Cinesina/metabolismo , Microtúbulos/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Katanina/metabolismo , Cinética , Doenças Neurodegenerativas/metabolismo
3.
Neuron ; 103(4): 642-657.e7, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31255486

RESUMO

Neuronal subtypes show diverse injury responses, but the molecular underpinnings remain elusive. Using transgenic mice that allow reliable visualization of axonal fate, we demonstrate that intrinsically photosensitive retinal ganglion cells (ipRGCs) are both resilient to cell death and highly regenerative. Using RNA sequencing (RNA-seq), we show genes that are differentially expressed in ipRGCs and that associate with their survival and axon regeneration. Strikingly, thrombospondin-1 (Thbs1) ranked as the most differentially expressed gene, along with the well-documented injury-response genes Atf3 and Jun. THBS1 knockdown in RGCs eliminated axon regeneration. Conversely, RGC overexpression of THBS1 enhanced regeneration in both ipRGCs and non-ipRGCs, an effect that was dependent on syndecan-1, a known THBS1-binding protein. All structural domains of the THBS1 were not equally effective; the trimerization and C-terminal domains promoted regeneration, while the THBS type-1 repeats were dispensable. Our results identify cell-type-specific induction of Thbs1 as a novel gene conferring high regenerative capacity.


Assuntos
Regeneração Nervosa/fisiologia , Células Ganglionares da Retina/fisiologia , Trombospondina 1/fisiologia , Animais , Apoptose , Axônios/metabolismo , Linhagem Celular , Feminino , Perfilação da Expressão Gênica , Genes Reporter , Fator de Crescimento Insulin-Like I/deficiência , Fator de Crescimento Insulin-Like I/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Compressão Nervosa , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/fisiopatologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Opsinas de Bastonetes/deficiência , Opsinas de Bastonetes/fisiologia , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/fisiologia , Trombospondina 1/biossíntese , Trombospondina 1/genética , Transcrição Genética
4.
Psychopharmacology (Berl) ; 236(9): 2761-2771, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31165206

RESUMO

RATIONALE: As the hub of memory and space, hippocampus is very sensitive to a wide variety of injuries and is one of the earliest brain structures to develop neurodegenerative changes in AD. Previous research has showed a protective effect of potassium 2-(l-hydroxypentyl)-benzoate (PHPB) on cognitive deficits in animal models of AD. However, it is unclear whether this protective effect is associated with hippocampal alterations. OBJECTIVES: The present study was conducted to evaluate the protective effect of PHPB on hippocampal neurodegenerative changes in middle-aged APP/PS1 mice. METHODS: Ten-month-old male APP/PS1 transgenic mice and age-matched wild-type mice were randomly divided into three groups. PHPB-treated APP/PS1 group received 30 mg/kg PHPB by oral gavage once daily for 12 weeks. Wild-type group and APP/PS1 group received the same volume of water alone. Twelve weeks later, mice (13-month-old) were tested for in vivo 1H-MRS examination and then sacrificed for subsequent biochemical and pathological examinations using transmission electron microscopy, Golgi staining, immunohistochemistry, and western blotting. RESULTS: We found that PHPB treatment significantly improved the micromorphology of hippocampal neurons and subcellular organelles, ameliorated synapse loss and presynaptic axonal dystrophy, increased hippocampal dendritic spine density and dendritic complexity, enhanced the expression of hippocampal synapse-associated proteins, and improved hippocampal metabolism in middle-aged APP/PS1 mice. CONCLUSIONS: Our study showed for the first time the protective effect of PHPB on hippocampal neurons, synapses, and dystrophic axons in APP/PS1 mice, which to some extent revealed the possible mechanism for its ability to improve cognition in animal models of AD.


Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Axônios/metabolismo , Hipocampo/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Presenilina-1/metabolismo , Sinapses/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Benzoatos/administração & dosagem , Cognição/efeitos dos fármacos , Cognição/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Pentanóis/administração & dosagem , Potássio/administração & dosagem , Presenilina-1/genética , Distribuição Aleatória , Sinapses/genética , Sinapses/patologia , Resultado do Tratamento
5.
Cell Mol Life Sci ; 76(16): 3141-3156, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31168660

RESUMO

Neurotrauma, a term referencing both traumatic brain and spinal cord injuries, is unique to neurodegeneration in that onset is clearly defined. From the perspective of matrix metalloproteinases (MMPs), there is opportunity to define their temporal participation in injury and recovery beginning at the level of the synapse. Here we examine the diverse roles of MMPs in the context of targeted insults (optic nerve lesion and hippocampal and olfactory bulb deafferentation), and clinically relevant focal models of traumatic brain and spinal cord injuries. Time-specific MMP postinjury signaling is critical to synaptic recovery after focal axonal injuries; members of the MMP family exhibit a signature temporal profile corresponding to axonal degeneration and regrowth, where they direct postinjury reorganization and synaptic stabilization. In both traumatic brain and spinal cord injuries, MMPs mediate early secondary pathogenesis including disruption of the blood-brain barrier, creating an environment that may be hostile to recovery. They are also critical players in wound healing including angiogenesis and the formation of an inhibitory glial scar. Experimental strategies to reduce their activity in the acute phase result in long-term neurological recovery after neurotrauma and have led to the first clinical trial in spinal cord injured pet dogs.


Assuntos
Metaloproteinases da Matriz/metabolismo , Traumatismos da Medula Espinal/patologia , Animais , Axônios/metabolismo , Barreira Hematoencefálica/metabolismo , Hipocampo/metabolismo , Humanos , Bulbo Olfatório/metabolismo , Nervo Óptico/metabolismo , Traumatismos da Medula Espinal/metabolismo , Sinapses/fisiologia
6.
Nat Methods ; 16(7): 615-618, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209383

RESUMO

We advance two-photon microscopy for near-diffraction-limited imaging up to 850 µm below the pia in awake mice. Our approach combines direct wavefront sensing of light from a guidestar (formed by descanned fluorescence from Cy5.5-conjugated dextran in brain microvessels) with adaptive optics to compensate for tissue-induced aberrations in the wavefront. We achieve high signal-to-noise ratios in recordings of glutamate release from thalamocortical axons and calcium transients in spines of layer 5b basal dendrites during active tactile sensing.


Assuntos
Axônios/metabolismo , Espinhas Dendríticas/metabolismo , Animais , Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Razão Sinal-Ruído
7.
PLoS Genet ; 15(6): e1007960, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31233487

RESUMO

UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions are consistent with RHO-1 and RHGF-1 acting with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Netrinas/genética , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/genética , Animais , Orientação de Axônios/genética , Axônios/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Movimento Celular/genética , Polaridade Celular/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Cones de Crescimento/metabolismo , Microtúbulos/genética , Fatores de Crescimento Neural/genética , Fenótipo , Pseudópodes/genética , Receptores de Superfície Celular/genética , Transdução de Sinais/genética
8.
Nat Commun ; 10(1): 2593, 2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31197139

RESUMO

Prion-like domains (PLDs), defined by their low sequence complexity and intrinsic disorder, are present in hundreds of human proteins. Although gain-of-function mutations in the PLDs of neuronal RNA-binding proteins have been linked to neurodegenerative disease progression, the physiological role of PLDs and their range of molecular functions are still largely unknown. Here, we show that the PLD of Drosophila Imp, a conserved component of neuronal ribonucleoprotein (RNP) granules, is essential for the developmentally-controlled localization of Imp RNP granules to axons and regulates in vivo axonal remodeling. Furthermore, we demonstrate that Imp PLD restricts, rather than promotes, granule assembly, revealing a novel modulatory function for PLDs in RNP granule homeostasis. Swapping the position of Imp PLD compromises RNP granule dynamic assembly but not transport, suggesting that these two functions are uncoupled. Together, our study uncovers a physiological function for PLDs in the spatio-temporal control of neuronal RNP assemblies.


Assuntos
Transporte Axonal/fisiologia , Grânulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Domínios Proteicos/fisiologia , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Linhagem Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Microscopia de Fluorescência , Modelos Animais , Príons/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
9.
Nat Cell Biol ; 21(6): 768-777, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061466

RESUMO

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.


Assuntos
Axônios/metabolismo , Mitocôndrias/genética , Fotólise , Multimerização Proteica/efeitos da radiação , Actinas/antagonistas & inibidores , Animais , Axônios/química , Axônios/efeitos da radiação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células COS , Cercopithecus aethiops , Cinesina/química , Luz , Microtúbulos/genética , Microtúbulos/efeitos da radiação , Mitocôndrias/química , Mitocôndrias/efeitos da radiação , Neurônios/química , Neurônios/efeitos da radiação , Polimerização/efeitos dos fármacos , Domínios Proteicos/genética , Domínios Proteicos/efeitos da radiação , Multimerização Proteica/genética , Sinapses/química , Sinapses/genética , Sinapses/efeitos da radiação , Proteínas de Ligação a Tacrolimo/química , Proteínas de Ligação a Tacrolimo/genética , Tiazolidinas/farmacologia , Proteína Vesicular 1 de Transporte de Glutamato/genética
10.
Cell Mol Life Sci ; 76(21): 4355-4368, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31041455

RESUMO

Axons in the central nervous system (CNS) typically fail to regenerate after injury. This failure is multi-factorial and caused in part by disruption of the axonal cytoskeleton. The cytoskeleton, in particular microtubules (MT), plays a critical role in axonal transport and axon growth during development. In this regard, members of the kinesin superfamily of proteins (KIFs) regulate the extension of primary axons toward their targets and control the growth of collateral branches. KIF2A negatively regulates axon growth through MT depolymerization. Using three different injury models to induce SCI in adult rats, we examined the temporal and cellular expression of KIF2A in the injured spinal cord. We observed a progressive increase of KIF2A expression with maximal levels at 10 days to 8 weeks post-injury as determined by Western blot analysis. KIF2A immunoreactivity was present in axons, spinal neurons and mature oligodendrocytes adjacent to the injury site. Results from the present study suggest that KIF2A at the injured axonal tips may contribute to neurite outgrowth inhibition after injury, and that its increased expression in inhibitory spinal neurons adjacent to the injury site might contribute to an intrinsic wiring-control mechanism associated with neuropathic pain. Further studies will determine whether KIF2A may be a potential target for the development of regeneration-promoting or pain-preventing therapies.


Assuntos
Cinesina/análise , Cinesina/metabolismo , Traumatismos da Medula Espinal/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Cinesina/genética , Masculino , Regeneração Nervosa/genética , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Medula Espinal/patologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia
11.
Invest Ophthalmol Vis Sci ; 60(6): 2274-2285, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31112612

RESUMO

Purpose: Retraction of the axon terminals of rod photoreceptors after retinal detachment breaks the first synapse in the visual pathway, resulting in visual impairment. Previous work showed that the mechanism of axonal retraction involves RhoA signaling and its downstream effector LIM Kinase (LIMK) activation. We examined the response of the downstream component cofilin, a direct binding protein of actin filaments, as well as the regulation by RhoA-LIMK-Cofilin signaling of actin assembly/disassembly, in the presynaptic ribbon terminal of injured rod cells. Methods: Injury was produced by retinal detachment or rod cell isolation. Detached porcine retina was probed for levels and localization of phosphorylated cofilin with Western blots and confocal microscopy, whereas rod cell cultures of dissociated salamander retina were examined for filamentous actin assembly/disassembly with a barbed end assay and phalloidin staining. Results: A detachment increased phosphorylation of cofilin in retinal explants; phosphorylation occurred in rod terminals in sections of detached retina. Isolation of rod cells resulted in axon retraction accompanied by an increase in actin barbed ends and a decrease in net filament labeling. All changes were significantly reduced by either Rho kinase (ROCK) or LIMK inhibition, using Y27632 or BMS-5, respectively. Cytochalasin D also reduced retraction and stabilized filaments in isolated rod cells. Conclusions: These results indicate that actin depolymerization via activation of RhoA downstream kinases and cofilin contributes to axon retraction. Preventing depolymerization, in addition to actomyosin contraction, may stabilize ribbon synapses after trauma.


Assuntos
Actinas/metabolismo , Cofilina 1/metabolismo , Retina/lesões , Descolamento Retiniano/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Amidas/farmacologia , Animais , Axônios/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Quinases Lim/antagonistas & inibidores , Plasticidade Neuronal/fisiologia , Piridinas/farmacologia , Suínos , Quinases Associadas a rho/farmacologia , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores
12.
Chem Biol Interact ; 308: 70-79, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31100276

RESUMO

Tri-ortho-cresyl phosphate (TOCP) is a typical organophosphorus compound that can cause organophosphate-induced delayed neuropathy (OPIDN), which is pathologically characterized by axonal degeneration. Nowadays, mitochondrial dysfunction is regarded as a potential mechanism contributing to OPIDN progress. Mitophagy, a selective type of autophagy, is required to segregate damaged mitochondria from healthy mitochondrial networks and deliver them to lysosome for degradation. This research was designed to investigate the role of mitophagy in axon degeneration following TOCP administration in an in vitro model. Differentiated neuro2a (N2a) cells were divided into four groups and treated with 0, 5, 10, and 20 µM TOCP for 24 h, respectively. The critical proteins in PINK1-Parkin-dependent mitophagy including LC3, P62, PINK1, Parkin, mitochondrial proteins, and autophagic receptors were detected by immunoblotting and immunofluorescence. After TOCP treatment, increased level of ROS in N2a cells revealed a significant mitochondria damage. Meanwhile, it was observed that much more PINK1, Parkin, and LC3-II were translocated to the mitochondria. Furthermore, immunofluorescence analysis demonstrated that the co-localization of Parkin and LC3 was significantly increased. These results suggested that PINK1-Parkin dependent mitophagy pathway in N2a cells was activated by TOCP treatment. In addition, P62, a major autophagic receptor, was markedly accumulated on the mitochondria, which indicated that P62 might play a critical role in facilitating mitophagy under TOCP-induced axonal degeneration. Taken together, our results suggest that TOCP exposure resulted in the activation of PINK1-Parkin-dependent mitophagy in N2a cells. Mitophagy may act as a positively reactive mode in eliminating dysfunctional mitochondria and therefore protect neurons against TOCP neurotoxicity.


Assuntos
Degradação Mitocondrial/efeitos dos fármacos , Proteínas Quinases/metabolismo , Tritolil Fosfatos/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína Sequestossoma-1/metabolismo
13.
Phys Rev E ; 99(2-1): 022408, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30934335

RESUMO

Geometrical cues play an essential role in neuronal growth. Here, we quantify axonal growth on surfaces with controlled geometries and report a general stochastic approach that quantitatively describes the motion of growth cones. We show that axons display a strong directional alignment on micropatterned surfaces when the periodicity of the patterns matches the dimension of the growth cone. The growth cone dynamics on surfaces with uniform geometry is described by a linear Langevin equation with both deterministic and stochastic contributions. In contrast, axonal growth on surfaces with periodic patterns is characterized by a system of two generalized Langevin equations with both linear and quadratic velocity dependence and stochastic noise. We combine experimental data with theoretical analysis to measure the key parameters of the growth cone motion: angular distributions, correlation functions, diffusion coefficients, characteristics speeds, and damping coefficients. We demonstrate that axonal dynamics displays a crossover from an Ornstein-Uhlenbeck process to a nonlinear stochastic regime when the geometrical periodicity of the pattern approaches the linear dimension of the growth cone. Growth alignment is determined by surface geometry, which is fully quantified by the deterministic part of the Langevin equation. These results provide insight into the role of curvature sensing proteins and their interactions with geometrical cues.


Assuntos
Neurônios/citologia , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Proliferação de Células/efeitos dos fármacos , Dimetilpolisiloxanos/farmacologia , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Nylons/farmacologia , Ratos
14.
Histochem Cell Biol ; 152(1): 35-45, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30976911

RESUMO

Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.


Assuntos
Axônios/metabolismo , Próstata/embriologia , Próstata/inervação , Animais , Axônios/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Próstata/citologia , Próstata/patologia
15.
Dev Growth Differ ; 61(4): 276-282, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30968390

RESUMO

Skin development is tightly temporally coordinated with its sensory innervation, which consists of the peripheral branches of the dorsal root ganglion (DRG) axons. Various studies suggest that the skin produces a long-range attractant for the sensory axons. However, the exact identity of the guidance cue(s) remains unclear. To reveal the detailed molecular mechanism that controls DRG axon guidance and targeting, manipulation of specific skin layers at specific time points are required. To test a variety of attractants that can be expressed in specific skin layers at specific timepoints, we combined in utero electroporation with the Tol2 transposon system to induce long-term transgene expression in the developing mouse skin, including in the highly proliferative epidermal stem cells (basal layer) and their descendants (spinous and granular layer cells). The plasmid solution was injected as close to the hindpaw plantar surface as possible. Immediately, electric pulses were passed through the embryo to transduce the plasmid DNA into hindpaw skin cells. Balancing outcome measurements including: embryo survival, transfection efficiency, and the efficiency of transgene integration into host cells, we found that IUE was best performed on E13.5, and using an electroporation voltage of 34V. After immunostaining embryonic and early postnatal skin tissue sections for keratinocyte and sensory axon markers, we observe the growth of axons into skin epidermal layers including areas expressing EGFP. Therefore, this method is useful for studying the interaction between axon growth and epidermal cell division/differentiation.


Assuntos
Epiderme/inervação , Epiderme/metabolismo , Neurônios/metabolismo , Pele/inervação , Pele/metabolismo , Transgenes/genética , Animais , Axônios/metabolismo , Células Epidérmicas/citologia , Células Epidérmicas/metabolismo , Epiderme/embriologia , Epiderme/crescimento & desenvolvimento , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Gravidez , Pele/embriologia , Pele/crescimento & desenvolvimento
16.
Acta Histochem ; 121(4): 383-391, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30846200

RESUMO

The claustrum is a bilateral subcortical nucleus situated between the insular cortex and the striatum in the brain of all mammals. It consists of two embryologically distinct subdivisions - dorsal and ventral claustrum. The claustrum has high connectivity with various areas of the cortex, subcortical and allocortical structures. It has long been suggested that the various claustral connections have different types of synaptic contacts at the claustral neurons. However, to the best of our knowledge, the literature data on the ultrastructural organization of the different types of synaptic contacts in the dorsal claustrum are very few. Therefore, the aim of our study was to observe and describe the synaptic organization of the dorsal claustrum in the cat. We used a total of 10 adult male cats and conducted an ultrastructural study under a transmission electron microscope as per established protocol. We described a multitude of dendritic spines, which were subdivided into two types - with and without foot processes. Based on the size and shape of the terminal boutons, the quantity and distribution of vesicles and the characteristic features of the active synaptic zone, we described six types of synaptic boutons, most of which formed asymmetrical synaptic contacts. Furthermore, we reported the presence of axo-dendritic, axo-somatic, dendro-dendritic and axo-axonal synapses. The former two likely represent the morphological substrate of the corticoclaustral pathway, while the remaining two types have the ultrastructural features of inhibitory synapses, likely forming a local inhibitory circuit in the claustrum. In conclusion, the present study shares new information about the neuropil of the claustrum and proposes a systematic classification of the types of synaptic boutons and contacts observed in the dorsal claustrum of the cat, thus supporting its key and complex role as a structure integrating various information within the brain.


Assuntos
Sinapses/ultraestrutura , Animais , Axônios/metabolismo , Axônios/ultraestrutura , Gânglios da Base/metabolismo , Gânglios da Base/ultraestrutura , Gatos , Dendritos/metabolismo , Dendritos/ultraestrutura , Masculino , Neurônios/metabolismo , Neurônios/ultraestrutura , Sinapses/metabolismo
17.
eNeuro ; 6(1)2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838324

RESUMO

Development of the nervous system relies on a balance between axon and dendrite growth and subsequent pruning and degeneration. The developmental degeneration of dorsal root ganglion (DRG) sensory axons has been well studied in part because it can be readily modeled by removing the trophic support by nerve growth factor (NGF) in vitro. We have recently reported that axonal fragmentation induced by NGF withdrawal is dependent on Ca2+, and here, we address the mechanism of Ca2+ entry required for developmental axon degeneration of mouse embryonic DRG neurons. Our results show that the transient receptor potential vanilloid family member 1 (TRPV1) cation channel plays a critical role mediating Ca2+ influx in DRG axons withdrawn from NGF. We further demonstrate that TRPV1 activation is dependent on reactive oxygen species (ROS) generation that is driven through protein kinase C (PKC) and NADPH oxidase (NOX)-dependent pathways that become active upon NGF withdrawal. These findings demonstrate novel mechanistic links between NGF deprivation, PKC activation, ROS generation, and TRPV1-dependent Ca2+ influx in sensory axon degeneration.


Assuntos
Axônios/metabolismo , Cálcio/metabolismo , Gânglios Espinais/embriologia , Gânglios Espinais/metabolismo , Degeneração Neural/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Cátions Bivalentes/metabolismo , Células Cultivadas , Gânglios Espinais/citologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADPH Oxidases/metabolismo , Fator de Crescimento Neural/metabolismo , Proteína Quinase C/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPV/genética
18.
Cell Mol Life Sci ; 76(11): 2093-2110, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30826859

RESUMO

Cellular protein quality control (PQC) plays a significant role in the maintenance of cellular homeostasis. Failure of PQC mechanism may lead to various neurodegenerative diseases due to accumulation of aberrant proteins. To avoid such fatal neuronal conditions PQC employs autophagy and ubiquitin proteasome system (UPS) to degrade misfolded proteins. Few quality control (QC) E3 ubiquitin ligases interplay an important role to specifically recognize misfolded proteins for their intracellular degradation. Leucine-rich repeat and sterile alpha motif-containing 1 (LRSAM1) is a really interesting new gene (RING) class protein that possesses E3 ubiquitin ligase activity with promising applications in PQC. LRSAM1 is also known as RING finger leucine repeat rich (RIFLE) or TSG 101-associated ligase (TAL). LRSAM1 has various cellular functions as it modulates the protein aggregation, endosomal sorting machinery and virus egress from the cells. Thus, this makes LRSAM1 interesting to study not only in protein conformational disorders such as neurodegeneration but also in immunological and other cancerous disorders. Furthermore, LRSAM1 interacts with both cellular protein degradation machineries and hence it can participate in maintenance of overall cellular proteostasis. Still, more research work on the quality control molecular functions of LRSAM1 is needed to comprehend its roles in various protein aggregatory diseases. Earlier findings suggest that in a mouse model of Charcot-Marie-Tooth (CMT) disease, lack of LRSAM1 functions sensitizes peripheral axons to degeneration. It has been observed that in CMT the patients retain dominant and recessive mutations of LRSAM1 gene, which encodes most likely a defective protein. However, still the comprehensive molecular pathomechanism of LRSAM1 in neuronal functions and neurodegenerative diseases is not known. The current article systematically represents the molecular functions, nature and detailed characterization of LRSAM1 E3 ubiquitin ligase. Here, we review emerging molecular mechanisms of LRSAM1 linked with neurobiological functions, with a clear focus on the mechanism of neurodegeneration and also on other diseases. Better understanding of LRSAM1 neurobiological and intracellular functions may contribute to develop promising novel therapeutic approaches, which can also propose new lines of molecular beneficial targets for various neurodegenerative diseases.


Assuntos
Proteínas do Tecido Nervoso/genética , Doenças Neurodegenerativas/genética , Nervos Periféricos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Axônios/metabolismo , Axônios/patologia , Regulação da Expressão Gênica , Humanos , Mutação , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Nervos Periféricos/patologia , Agregados Proteicos , Dobramento de Proteína , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise , Proteostase/genética , Transdução de Sinais , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
19.
Nat Commun ; 10(1): 1414, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926781

RESUMO

Information processing by brain circuits depends on Ca2+-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca2+ in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca2+ indicator Cal-590 is Ca2+-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca2+ kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca2+ or spike-evoked Ca2+ entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca2+ entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.


Assuntos
Cálcio/metabolismo , Ácido Glutâmico/metabolismo , Homeostase , Imagem Tridimensional , Terminações Pré-Sinápticas/metabolismo , Animais , Axônios/metabolismo , Microscopia de Fluorescência , Nanopartículas/química , Plasticidade Neuronal , Fótons , Ratos Sprague-Dawley
20.
Nat Commun ; 10(1): 1331, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902970

RESUMO

GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aß, a component of senile plaques in Alzheimer's disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aß formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer's disease increases Aß formation.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Amiloide/metabolismo , Transporte Axonal , Receptores de GABA-B/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Dendritos/metabolismo , Epitopos/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Cinesina/metabolismo , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Estabilidade Proteica , Proteômica , Transdução de Sinais , Sinapses/metabolismo
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