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1.
Acta Neuropathol Commun ; 9(1): 180, 2021 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-34749824

RESUMEN

Pathologic inclusions composed of α-synuclein called Lewy pathology are hallmarks of Parkinson's Disease (PD). Dominant inherited mutations in leucine rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. Lewy pathology is found in the majority of individuals with LRRK2-PD, particularly those with the G2019S-LRRK2 mutation. Lewy pathology in LRRK2-PD associates with increased non-motor symptoms such as cognitive deficits, anxiety, and orthostatic hypotension. Thus, understanding the relationship between LRRK2 and α-synuclein could be important for determining the mechanisms of non-motor symptoms. In PD models, expression of mutant LRRK2 reduces membrane localization of α-synuclein, and enhances formation of pathologic α-synuclein, particularly when synaptic activity is increased. α-Synuclein and LRRK2 both localize to the presynaptic terminal. LRRK2 plays a role in membrane traffic, including axonal transport, and therefore may influence α-synuclein synaptic localization. This study shows that LRRK2 kinase activity influences α-synuclein targeting to the presynaptic terminal. We used the selective LRRK2 kinase inhibitors, MLi-2 and PF-06685360 (PF-360) to determine the impact of reduced LRRK2 kinase activity on presynaptic localization of α-synuclein. Expansion microscopy (ExM) in primary hippocampal cultures and the mouse striatum, in vivo, was used to more precisely resolve the presynaptic localization of α-synuclein. Live imaging of axonal transport of α-synuclein-GFP was used to investigate the impact of LRRK2 kinase inhibition on α-synuclein axonal transport towards the presynaptic terminal. Reduced LRRK2 kinase activity increases α-synuclein overlap with presynaptic markers in primary neurons, and increases anterograde axonal transport of α-synuclein-GFP. In vivo, LRRK2 inhibition increases α-synuclein overlap with glutamatergic, cortico-striatal terminals, and dopaminergic nigral-striatal presynaptic terminals. The findings suggest that LRRK2 kinase activity plays a role in axonal transport, and presynaptic targeting of α-synuclein. These data provide potential mechanisms by which LRRK2-mediated perturbations of α-synuclein localization could cause pathology in both LRRK2-PD, and idiopathic PD.

3.
Commun Biol ; 4(1): 1140, 2021 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-34588600

RESUMEN

Parkinson's disease (PD) is a chronic neurological disorder associated with the misfolding of alpha-synuclein (α-syn) into aggregates within nerve cells that contribute to their neurodegeneration. Recent evidence suggests α-syn aggregation may begin in the gut and travel to the brain along the vagus nerve, with microbes potentially a trigger initiating α-syn misfolding. However, the effects α-syn alterations on the gut virome have not been investigated. In this study, we show longitudinal faecal virome changes in rats administered either monomeric or preformed fibrils (PFF) of α-syn directly into their enteric nervous system. Differential changes in rat viromes were observed when comparing monomeric and PFF α-syn, with alterations compounded by the addition of LPS. Changes in rat faecal viromes were observed after one month and did not resolve within the study's five-month observational period. These results suggest that virome alterations may be reactive to host α-syn changes that are associated with PD development.

6.
eNeuro ; 8(3)2021.
Artículo en Inglés | MEDLINE | ID: mdl-33972291

RESUMEN

Synucleinopathies including Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by neuronal intracellular inclusions of α-synuclein. PD dementia (PDD) and DLB are collectively the second most common cause of neurodegenerative dementia. In addition to associated inclusions, Lewy body diseases (LBDs) have dopaminergic neurodegeneration, motor defects and cognitive changes. The microtubule-associated protein tau has been implicated in LBDs, but the exact role of the protein and how it influences formation of α-synuclein inclusions is unknown. Reducing endogenous tau levels is protective in multiple models of Alzheimer's disease (AD), tauopathies, and in some transgenic synucleinopathy mouse models. Recombinant α-synuclein and tau proteins interact in vitro Here, we show tau and α-synuclein colocalize at excitatory presynaptic terminals. However, tau heterozygous and tau knock-out mice do not show a reduction in fibril-induced α-synuclein inclusions formation in primary cortical neurons, or after intrastriatal injections of fibrils at 1.5 month or six months later. At six months following intrastriatal injections, wild-type, tau heterozygous and tau knock-out mice showed a 50% reduction in dopamine neurons in the substantia nigra pars compacta (SNc) compared with mice injected with α-synuclein monomer, but there were no statistically significant differences across genotypes. These data suggest the role of tau in the pathogenesis of LBDs is distinct from AD, and Lewy pathology formation may be independent of endogenous tau.


Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Animales , Neuronas Dopaminérgicas , Ratones , Ratones Transgénicos , alfa-Sinucleína/genética , Proteínas tau/genética
7.
Front Cell Neurosci ; 15: 626128, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33737866

RESUMEN

Parkinson's disease (PD) is a progressive neurodegenerative disease that impairs movement as well as causing multiple other symptoms such as autonomic dysfunction, rapid eye movement (REM) sleep behavior disorder, hyposmia, and cognitive changes. Loss of dopamine neurons in the substantia nigra pars compacta (SNc) and loss of dopamine terminals in the striatum contribute to characteristic motor features. Although therapies ease the symptoms of PD, there are no treatments to slow its progression. Accumulating evidence suggests that synaptic impairments and axonal degeneration precede neuronal cell body loss. Early synaptic changes may be a target to prevent disease onset and slow progression. Imaging of PD patients with radioligands, post-mortem pathologic studies in sporadic PD patients, and animal models of PD demonstrate abnormalities in presynaptic terminals as well as postsynaptic dendritic spines. Dopaminergic and excitatory synapses are substantially reduced in PD, and whether other neuronal subtypes show synaptic defects remains relatively unexplored. Genetic studies implicate several genes that play a role at the synapse, providing additional support for synaptic dysfunction in PD. In this review article we: (1) provide evidence for synaptic defects occurring in PD before neuron death; (2) describe the main genes implicated in PD that could contribute to synapse dysfunction; and (3) show correlations between the expression of Snca mRNA and mouse homologs of PD GWAS genes demonstrating selective enrichment of Snca and synaptic genes in dopaminergic, excitatory and cholinergic neurons. Altogether, these findings highlight the need for novel therapeutics targeting the synapse and suggest that future studies should explore the roles for PD-implicated genes across multiple neuron types and circuits.

8.
Mol Neurodegener ; 15(1): 19, 2020 03 06.
Artículo en Inglés | MEDLINE | ID: mdl-32143659

RESUMEN

The two main pathological hallmarks of Parkinson's disease are loss of dopamine neurons in the substantia nigra pars compacta and proteinaceous amyloid fibrils composed mostly of α-synuclein, called Lewy pathology. Levodopa to enhance dopaminergic transmission remains one of the most effective treatment for alleviating the motor symptoms of Parkinson's disease (Olanow, Mov Disord 34:812-815, 2019). In addition, deep brain stimulation (Bronstein et al., Arch Neurol 68:165, 2011) to modulate basal ganglia circuit activity successfully alleviates some motor symptoms. MRI guided focused ultrasound in the subthalamic nucleus is a promising therapeutic strategy as well (Martinez-Fernandez et al., Lancet Neurol 17:54-63, 2018). However, to date, there exists no treatment that stops the progression of this disease. The findings that α-synuclein can be released from neurons and inherited through interconnected neural networks opened the door for discovering novel treatment strategies to prevent the formation and spread of Lewy pathology with the goal of halting PD in its tracks. This hypothesis is based on discoveries that pathologic aggregates of α-synuclein induce the endogenous α-synuclein protein to adopt a similar pathologic conformation, and is thus self-propagating. Phase I clinical trials are currently ongoing to test treatments such as immunotherapy to prevent the neuron to neuron spread of extracellular aggregates. Although tremendous progress has been made in understanding how Lewy pathology forms and spreads throughout the brain, cell intrinsic factors also play a critical role in the formation of pathologic α-synuclein, such as mechanisms that increase endogenous α-synuclein levels, selective expression profiles in distinct neuron subtypes, mutations and altered function of proteins involved in α-synuclein synthesis and degradation, and oxidative stress. Strategies that prevent the formation of pathologic α-synuclein should consider extracellular release and propagation, as well as neuron intrinsic mechanisms.


Asunto(s)
Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , alfa-Sinucleína/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Progresión de la Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Humanos , Agregado de Proteínas/fisiología
9.
Nat Neurosci ; 23(3): 327-336, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32066981

RESUMEN

Parkinson's disease is a synucleinopathy that is characterized by motor dysfunction, death of midbrain dopaminergic neurons and accumulation of α-synuclein (α-Syn) aggregates. Evidence suggests that α-Syn aggregation can originate in peripheral tissues and progress to the brain via autonomic fibers. We tested this by inoculating the duodenal wall of mice with α-Syn preformed fibrils. Following inoculation, we observed gastrointestinal deficits and physiological changes to the enteric nervous system. Using the AAV-PHP.S capsid to target the lysosomal enzyme glucocerebrosidase for peripheral gene transfer, we found that α-Syn pathology is reduced due to the increased expression of this protein. Lastly, inoculation of α-Syn fibrils in aged mice, but not younger mice, resulted in progression of α-Syn histopathology to the midbrain and subsequent motor defects. Our results characterize peripheral synucleinopathy in prodromal Parkinson's disease and explore cellular mechanisms for the gut-to-brain progression of α-Syn pathology.


Asunto(s)
Encéfalo/patología , Enfermedades del Sistema Digestivo/patología , Sinucleinopatías/metabolismo , Sinucleinopatías/patología , Animales , Duodeno/patología , Sistema Nervioso Entérico/patología , Glucosilceramidasa/biosíntesis , Glucosilceramidasa/genética , Mesencéfalo/patología , Ratones , Ratones Endogámicos C57BL , Trastornos del Movimiento/etiología , Trastornos del Movimiento/patología , Fibras Nerviosas/patología , Nocicepción , Ganglio Nudoso/patología
11.
Neurobiol Dis ; 134: 104708, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31837424

RESUMEN

Parkinson's disease (PD) is defined by motor symptoms such as tremor at rest, bradykinesia, postural instability, and stiffness. In addition to the classical motor defects that define PD, up to 80% of patients experience cognitive changes and psychiatric disturbances, referred to as PD dementia (PDD). Pathologically, PD is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and intracellular inclusions, called Lewy bodies and Lewy neurites, composed mostly of α-synuclein. Much of PD research has focused on the role of α-synuclein aggregates in degeneration of SNpc dopamine neurons because of the impact of loss of striatal dopamine on the classical motor phenotypes. However, abundant Lewy pathology is also found in other brain regions including the cortex and limbic brain regions such as the amygdala, which may contribute to non-motor phenotypes. Little is known about the consequences of α-synuclein inclusions in these brain regions, or in neuronal subtypes other than dopamine neurons. This project expands knowledge on how α-synuclein inclusions disrupt behavior, specifically non-motor symptoms of synucleinopathies. We show that bilateral injections of fibrils into the striatum results in robust bilateral α-synuclein inclusion formation in the cortex and amygdala. Inclusions in the amygdala and prefrontal cortex primarily localize to excitatory neurons, but unbiased stereology shows no significant loss of neurons in the amygdala or cortex. Fibril injected mice show defects in a social dominance behavioral task and fear conditioning, tasks that are associated with prefrontal cortex and amygdala function. Together, these observations suggest that seeded α-synuclein inclusion formation impairs behaviors associated with cortical and amygdala function, without causing cell loss, in brain areas that may play important roles in the complex cognitive features of PDD.


Asunto(s)
Amígdala del Cerebelo/patología , Conducta Animal/fisiología , Corteza Cerebral/patología , Cuerpos de Inclusión/patología , alfa-Sinucleína/metabolismo , Amígdala del Cerebelo/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Corteza Cerebral/metabolismo , Condicionamiento Clásico , Cuerpo Estriado/efectos de los fármacos , Femenino , Cuerpos de Inclusión/metabolismo , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Enfermedad de Parkinson/psicología , Prueba de Desempeño de Rotación con Aceleración Constante , alfa-Sinucleína/administración & dosificación
12.
Mov Disord ; 34(10): 1406-1422, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31483900

RESUMEN

While current effective therapies are available for the symptomatic control of PD, treatments to halt the progressive neurodegeneration still do not exist. Loss of dopamine neurons in the SNc and dopamine terminals in the striatum drive the motor features of PD. Multiple lines of research point to several pathways which may contribute to dopaminergic neurodegeneration. These pathways include extensive axonal arborization, mitochondrial dysfunction, dopamine's biochemical properties, abnormal protein accumulation of α-synuclein, defective autophagy and lysosomal degradation, and synaptic impairment. Thus, understanding the essential features and mechanisms of dopaminergic neuronal vulnerability is a major scientific challenge and highlights an outstanding need for fostering effective therapies against neurodegeneration in PD. This article, which arose from the Movement Disorders 2018 Conference, discusses and reviews the possible mechanisms underlying neuronal vulnerability and potential therapeutic approaches in PD. © 2019 International Parkinson and Movement Disorder Society.


Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Enfermedad de Parkinson/fisiopatología , Trastornos Parkinsonianos/fisiopatología , Terminales Presinápticos/metabolismo , Animales , Axones/metabolismo , Emparejamiento Cromosómico/fisiología , Humanos
13.
J Vis Exp ; (148)2019 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-31205308

RESUMEN

Use of the in vivo alpha-synuclein preformed fibril (α-syn PFF) model of synucleinopathy is gaining popularity among researchers aiming to model Parkinson's disease synucleinopathy and nigrostriatal degeneration. The standardization of α-syn PFF generation and in vivo application is critical in order to ensure consistent, robust α-syn pathology. Here, we present a detailed protocol for the generation of fibrils from monomeric α-syn, post-fibrilization quality control steps, and suggested parameters for successful neurosurgical injection of α-syn PFFs into rats or mice. Starting with monomeric α-syn, fibrilization occurs over a 7-day incubation period while shaking at optimal buffer conditions, concentration, and temperature. Post-fibrilization quality control is assessed by the presence of pelletable fibrils via sedimentation assay, the formation of amyloid conformation in the fibrils with a thioflavin T assay, and electron microscopic visualization of the fibrils. Whereas successful validation using these assays is necessary for success, they are not sufficient to guarantee PFFs will seed α-syn inclusions in neurons, as such aggregation activity of each PFF batch should be tested in cell culture or in pilot animal cohorts. Prior to use, PFFs must be sonicated under precisely standardized conditions, followed by examination using electron microscopy or dynamic light scattering to confirm fibril lengths are within optimal size range, with an average length of 50 nm. PFFs can then be added to cell culture media or used in animals. Pathology detectable by immunostaining for phosphorylated α-syn (psyn; serine 129) is apparent days or weeks later in cell culture and rodent models, respectively.


Asunto(s)
Neuronas/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Células Cultivadas , Ratones , Enfermedad de Parkinson , Ratas , Sinucleinopatías
14.
J Biol Chem ; 294(27): 10392-10406, 2019 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-31142553

RESUMEN

Parkinson's disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated α-synuclein (α-syn). These inclusions are associated with behavioral and pathological PD phenotypes. One strategy for therapeutic interventions is to prevent the formation of these inclusions to halt disease progression. α-Synuclein exists in multiple structural forms, including disordered, nonamyloid oligomers, ordered amyloid oligomers, and fibrils. It is critical to understand which conformers contribute to specific PD phenotypes. Here, we utilized a mouse model to explore the pathological effects of stable ß-amyloid-sheet oligomers compared with those of fibrillar α-synuclein. We biophysically characterized these species with transmission EM, atomic-force microscopy, CD spectroscopy, FTIR spectroscopy, analytical ultracentrifugation, and thioflavin T assays. We then injected these different α-synuclein forms into the mouse striatum to determine their ability to induce PD-related phenotypes. We found that ß-sheet oligomers produce a small but significant loss of dopamine neurons in the substantia nigra pars compacta (SNc). Injection of small ß-sheet fibril fragments, however, produced the most robust phenotypes, including reduction of striatal dopamine terminals, SNc loss of dopamine neurons, and motor-behavior defects. We conclude that although the ß-sheet oligomers cause some toxicity, the potent effects of the short fibrillar fragments can be attributed to their ability to recruit monomeric α-synuclein and spread in vivo and hence contribute to the development of PD-like phenotypes. These results suggest that strategies to reduce the formation and propagation of ß-sheet fibrillar species could be an important route for therapeutic intervention in PD and related disorders.


Asunto(s)
Enfermedad de Parkinson/patología , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Masculino , Ratones , Enfermedad de Parkinson/metabolismo , Fenotipo , Agregado de Proteínas , Conformación Proteica en Lámina beta , alfa-Sinucleína/química , alfa-Sinucleína/farmacología
15.
Methods Mol Biol ; 1948: 1-14, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30771165

RESUMEN

Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are characterized by intracellular inclusions composed mostly of α-synuclein (Baba et al., Am J Pathol 152:879-884, 1998). How inclusion formation impacts neuronal function prior to death is key to understanding disease progression and identifying therapeutic windows. In the α-synuclein fibril model, exposure of primary neurons to α-synuclein fibrils induces endogenously expressed α-synuclein to form inclusions which closely resembles pathologic mechanisms in humans with PD and DLB (Volpicelli-Daley et al., Neuron 72, 57-71, 2011). In this model, at 7 days after exposure of neurons to fibrils, when there is no neuron death, inclusions in the axon selectively impair axonal transport of endosomes carrying the TrkB receptor and LC3-positive autophagosomes (Volpicelli-Daley et al., Mol Biol Cell 25:4010-4023, 2014). In addition, the frequency and amplitude of spontaneous Ca2+ transients are reduced in neurons 7 days after fibril exposure. Here we discuss protocols for plating primary hippocampal neurons, generating fibrils and measuring axonal transport and Ca2+ transients. These assays provide additional assays of neurotoxicity allowing researchers to determine if a therapeutic intervention can prevent neuronal defects before intractable neurodegeneration.


Asunto(s)
Cuerpos de Inclusión/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Transporte Axonal , Calcio/metabolismo , Células Cultivadas , Femenino , Cuerpos de Inclusión/patología , Ratones , Imagen Molecular , Neuronas/patología , Embarazo , Agregado de Proteínas , Agregación Patológica de Proteínas , Células Piramidales/metabolismo
16.
Neurobiol Dis ; 124: 248-262, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30472299

RESUMEN

We recently identified a truncated and phosphorylated form of α-synuclein, pα-syn*, as a key neurotoxic α-synuclein species found in cultured neurons, as well as in mouse and Parkinson's disease patients' brains. Small pα-syn* aggregates localize to mitochondria and induce mitochondrial damage and fragmentation. Herein, we investigated the molecular basis of pα-syn*-induced toxicity. By immunofluorescence, we found phosphorylated MKK4, JNK, ERK5 and p38 MAPKs in pα-syn* inclusions. pJNK colocalized with pα-syn* at mitochondria and mitochondria-associated ER membranes where it was associated with BiP and pACC1, markers for the ER and energy deprivation, respectively. We also found that pα-syn* aggregates are tightly associated with small ptau aggregates of similar size. Pα-syn*/ptau inclusions localized to areas of mitochondrial damage and to mitophagic vesicles, showing their role in mitochondrial toxicity, mitophagy induction and their removal along with damaged mitochondrial fragments. Several MAPKs may act cooperatively to phosphorylate tau, notably JNK, p38 and GSK3ß, a non-MAPK that was also found phosphorylated in the vicinity of pα-syn*/ptau aggregates. These results add insight into the mechanisms by which pα-syn* exerts its toxic effects that include the phosphorylation of several kinases of the MAPK pathway, as well as the formation of ptau at the mitochondrial membrane, likely contributing to mitotoxicity. Thus pα-syn* appears to be the trigger of a series of kinase mediated pathogenic events and a link between α-syn pathology and tau, another protein known to aggregate in Parkinson's disease and other synucleinopathies.


Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Mitocondrias/metabolismo , Enfermedades Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Activación Enzimática/fisiología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/patología , Enfermedades Neurodegenerativas/patología , Enfermedades Neurodegenerativas/fisiopatología , Fosforilación
17.
J Neurosci ; 38(38): 8211-8232, 2018 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-30093536

RESUMEN

α-Synuclein (αsyn) is the key protein that forms neuronal aggregates in the neurodegenerative disorders Parkinson's disease (PD) and dementia with Lewy bodies. Recent evidence points to the prion-like spread of αsyn from one brain region to another. Propagation of αsyn is likely dependent on release, uptake, and misfolding. Under normal circumstances, this highly expressed brain protein functions normally without promoting pathology, yet the underlying endogenous mechanisms that prevent αsyn spread are not understood. 14-3-3 proteins are highly expressed brain proteins that have chaperone function and regulate protein trafficking. In this study, we investigated the potential role of the 14-3-3 proteins in the regulation of αsyn spread using two models of αsyn spread. In a paracrine αsyn model, 14-3-3θ promoted release of αsyn complexed with 14-3-3θ. Despite higher amounts of released αsyn, extracellular αsyn showed reduced oligomerization and seeding capability, reduced internalization, and reduced toxicity in primary mixed-gender mouse neurons. 14-3-3 inhibition reduced the amount of αsyn released, yet released αsyn was more toxic and demonstrated increased oligomerization, seeding capability, and internalization. In the preformed fibril model, 14-3-3 θ reduced αsyn aggregation and neuronal death, whereas 14-3-3 inhibition enhanced αsyn aggregation and neuronal death in primary mouse neurons. 14-3-3s blocked αsyn spread to distal chamber neurons not exposed directly to fibrils in multichamber, microfluidic devices. These findings point to 14-3-3s as a direct regulator of αsyn propagation, and suggest that dysfunction of 14-3-3 function may promote αsyn pathology in PD and related synucleinopathies.SIGNIFICANCE STATEMENT Transfer of misfolded aggregates of α-synuclein from one brain region to another is implicated in the pathogenesis of Parkinson's disease and other synucleinopathies. This process is dependent on active release, internalization, and misfolding of α-synuclein. 14-3-3 proteins are highly expressed chaperone proteins that interact with α-synuclein and regulate protein trafficking. We used two different models in which toxicity is associated with cell-to-cell transfer of α-synuclein to test whether 14-3-3s impact α-synuclein toxicity. We demonstrate that 14-3-3θ reduces α-synuclein transfer and toxicity by inhibiting oligomerization, seeding capability, and internalization of α-synuclein, whereas 14-3-3 inhibition accelerates the transfer and toxicity of α-synuclein in these models. Dysfunction of 14-3-3 function may be a critical mechanism by which α-synuclein propagation occurs in disease.


Asunto(s)
Proteínas 14-3-3/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Encéfalo/patología , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Enfermedad por Cuerpos de Lewy/metabolismo , Enfermedad por Cuerpos de Lewy/patología , Ratones , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Transporte de Proteínas/fisiología
18.
J Comp Neurol ; 526(12): 1978-1990, 2018 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-29888794

RESUMEN

α-Synuclein (α-syn) is an abundant presynaptic protein that is the primary constituent of inclusions that define Lewy body diseases (LBDs). In these inclusions, α-syn is phosphorylated at the serine-129 residue. Antibodies directed to this phosphorylation site are used to measure inclusion abundance and stage disease progression in preclinical models as well as in postmortem tissues in LBDs. While it is critical to reliably identify inclusions, phospho-specific antibodies often cross-react with nonspecific antigens. Four commercially available monoclonal antibodies, two from rabbits (clones EP1536Y and MJF-R13) and two from mice (81a and pSyn#64), have been the most widely used in detecting pS129-α-syn inclusions. Here, we systematically evaluated these antibodies in brain sections and protein lysates from rats and mice. All antibodies detected pS129-α-syn inclusions in the brain that were induced by preformed α-syn fibrils in wild-type rats and mice. Antibody titrations revealed that clones EP1536Y and 81a comparably labeled inclusions in both the perikarya and neuronal processes in contrast to clones MJF-R13 and pSyn#64 that incompletely labeled inclusions at various antibody concentrations. Except for EP1536Y, the clones produced nonspecific diffuse neuropil labeling in α-syn knockout mice as well as mice and rats injected with monomeric α-syn, with some nonspecific staining titrating with pS129-α-syn inclusions. By immunoblot, all the clones cross-reacted with proteins other than α-syn, warranting caution in interpretations of specificity. Clone EP1536Y uniquely and robustly detected endogenous pS129-α-syn in highly soluble protein fractions from the mouse brain. In summary, EP1536Y had the highest sensitivity and specificity for detecting pS129-α-syn.


Asunto(s)
Anticuerpos Monoclonales , Especificidad de Anticuerpos , alfa-Sinucleína/análisis , Animales , Anticuerpos Monoclonales/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , alfa-Sinucleína/inmunología
19.
Acta Neuropathol Commun ; 6(1): 35, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29716652

RESUMEN

Neuronal inclusions composed of α-synuclein (α-syn) characterize Parkinson's Disease (PD) and Dementia with Lewy bodies (DLB). Cognitive dysfunction defines DLB, and up to 80% of PD patients develop dementia. α-Syn inclusions are abundant in the hippocampus, yet functional consequences are unclear. To determine if pathologic α-syn causes neuronal defects, we induced endogenous α-syn to form inclusions resembling those found in diseased brains by treating hippocampal neurons with α-syn fibrils. At seven days after adding fibrils, α-syn inclusions are abundant in axons, but there is no cell death at this time point, allowing us to assess for potential alterations in neuronal function that are not caused by neuron death. We found that exposure of neurons to fibrils caused a significant reduction in mushroom spine densities, adding to the growing body of literature showing that altered spine morphology is a major pathologic phenotype in synucleinopathies. The reduction in spine densities occurred only in wild type neurons and not in neurons from α-syn knockout mice, suggesting that the changes in spine morphology result from fibril-induced corruption of endogenously expressed α-syn. Paradoxically, reduced postsynaptic spine density was accompanied by increased frequency of miniature excitatory postsynaptic currents (EPSCs) and presynaptic docked vesicles, suggesting enhanced presynaptic function. Action-potential dependent activity was unchanged, suggesting compensatory mechanisms responding to synaptic defects. Although activity at the level of the synapse was unchanged, neurons exposed to α-syn fibrils, showed reduced frequency and amplitudes of spontaneous Ca2+ transients. These findings open areas of research to determine the mechanisms that alter neuronal function in brain regions critical for cognition at time points before neuron death.


Asunto(s)
Hipocampo/citología , Neuronas/efectos de los fármacos , alfa-Sinucleína/toxicidad , Animales , Calcio/metabolismo , Células Cultivadas , Endotoxinas/toxicidad , Antagonistas del GABA/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Técnicas de Placa-Clamp , Fosfopiruvato Hidratasa/metabolismo , Picrotoxina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Potenciales Sinápticos/efectos de los fármacos , Tetrodotoxina/farmacología , Transducción Genética , alfa-Sinucleína/metabolismo
20.
Proc Natl Acad Sci U S A ; 115(11): E2634-E2643, 2018 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-29487216

RESUMEN

Exposure of cultured primary neurons to preformed α-synuclein fibrils (PFFs) leads to the recruitment of endogenous α-synuclein and its templated conversion into fibrillar phosphorylated α-synuclein (pα-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. Pα-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated α-syn species that we named "pα-syn*." We uniquely describe the existence of pα-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that pα-syn* results from incomplete autophagic degradation of pα-synF. Pα-synF was decorated with autophagic markers, but pα-syn* was not. Western blots revealed that pα-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, pα-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. Pα-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. Pα-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. Pα-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that pα-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating pα-syn* as a key neurotoxic α-syn species and a therapeutic target.


Asunto(s)
Autofagia/efectos de los fármacos , Mitofagia/efectos de los fármacos , Neurotoxinas , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína , Acetil-CoA Carboxilasa/química , Acetil-CoA Carboxilasa/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Química Encefálica , Técnicas de Cultivo de Célula , Células Cultivadas , Humanos , Lisosomas/metabolismo , Ratones , Mitocondrias , Neurotoxinas/química , Neurotoxinas/metabolismo , Neurotoxinas/toxicidad , Estrés Oxidativo/efectos de los fármacos , Fosforilación , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidad
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