RESUMEN

Synapses in the brain exhibit cell-type-specific differences in basal synaptic transmission and plasticity. Here, we evaluated cell-type-specific specializations in the composition of glutamatergic synapses, identifying Btbd11 as an inhibitory interneuron-specific, synapse-enriched protein. Btbd11 is highly conserved across species and binds to core postsynaptic proteins, including Psd-95. Intriguingly, we show that Btbd11 can undergo liquid-liquid phase separation when expressed with Psd-95, supporting the idea that the glutamatergic postsynaptic density in synapses in inhibitory interneurons exists in a phase-separated state. Knockout of Btbd11 decreased glutamatergic signaling onto parvalbumin-positive interneurons. Further, both in vitro and in vivo, Btbd11 knockout disrupts network activity. At the behavioral level, Btbd11 knockout from interneurons alters exploratory behavior, measures of anxiety, and sensitizes mice to pharmacologically induced hyperactivity following NMDA receptor antagonist challenge. Our findings identify a cell-type-specific mechanism that supports glutamatergic synapse function in inhibitory interneurons-with implications for circuit function and animal behavior.

Asunto(s)

Sinapsis , Transmisión Sináptica , Animales , Ratones , Homólogo 4 de la Proteína Discs Large/metabolismo , Interneuronas/metabolismo , Ratones Noqueados , Células Piramidales/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Factores de Transcripción/metabolismo

RESUMEN

OBJECTIVE: Dual leucine zipper kinase (DLK), which regulates the c-Jun N-terminal kinase pathway involved in axon degeneration and apoptosis following neuronal injury, is a potential therapeutic target in amyotrophic lateral sclerosis (ALS). This first-in-human study investigated safety, tolerability, and pharmacokinetics (PK) of oral GDC-0134, a small-molecule DLK inhibitor. Plasma neurofilament light chain (NFL) levels were explored in GDC-0134-treated ALS patients and DLK conditional knockout (cKO) mice. METHODS: The study included placebo-controlled, single and multiple ascending-dose (SAD; MAD) stages, and an open-label safety expansion (OLE) with adaptive dosing for up to 48 weeks. RESULTS: Forty-nine patients were enrolled. GDC-0134 (up to 1200 mg daily) was well tolerated in the SAD and MAD stages, with no serious adverse events (SAEs). In the OLE, three study drug-related SAEs occurred: thrombocytopenia, dysesthesia (both Grade 3), and optic ischemic neuropathy (Grade 4); Grade ≤2 sensory neurological AEs led to dose reductions/discontinuations. GDC-0134 exposure was dose-proportional (median half-life = 84 h). Patients showed GDC-0134 exposure-dependent plasma NFL elevations; DLK cKO mice also exhibited plasma NFL compared to wild-type littermates. INTERPRETATION: This trial characterized GDC-0134 safety and PK, but no adequately tolerated dose was identified. NFL elevations in GDC-0134-treated patients and DLK cKO mice raised questions about interpretation of biomarkers affected by both disease and on-target drug effects. The safety profile of GDC-0134 was considered unacceptable and led to discontinuation of further drug development for ALS. Further work is necessary to understand relationships between neuroprotective and potentially therapeutic effects of DLK knockout/inhibition and NFL changes in patients with ALS.

Asunto(s)

Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Proteínas de Neurofilamentos/sangre , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/efectos adversos , Adulto , Anciano , Esclerosis Amiotrófica Lateral/sangre , Animales , Biomarcadores/sangre , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Humanos , Quinasas Quinasa Quinasa PAM/deficiencia , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Evaluación de Resultado en la Atención de Salud , Inhibidores de Proteínas Quinasas/farmacocinética

RESUMEN

Alzheimer's disease (AD) is the most common form of dementia and the 6th leading cause of death in the US. The neuropathological hallmarks of the disease are extracellular amyloid-ß (Aß) plaques and intraneuronal hyperphosphorylated tau aggregates. Genetic variants of TREM2 (triggering receptor expressed on myeloid cells 2), a cell-surface receptor expressed selectively in myeloid cells, greatly increase the risk of AD, implicating microglia and the innate immune system as pivotal factors in AD pathogenesis. Recent studies have advanced our understanding of TREM2 biology and microglial activities in aging and neurodegenerative brains, providing new insights into TREM2 functions in amyloid plaque maintenance, microglial envelopment of plaque, microglia viability, and the identification of novel TREM2 ligands. Our increased understanding of TREM2 and microglia has opened new avenues for therapeutic intervention to delay or prevent the progression of AD.

Asunto(s)

Envejecimiento/inmunología , Enfermedad de Alzheimer/inmunología , Inmunidad Innata , Glicoproteínas de Membrana/inmunología , Microglía/inmunología , Células Mieloides/inmunología , Receptores Inmunológicos/inmunología , Envejecimiento/genética , Envejecimiento/patología , Enfermedad de Alzheimer/epidemiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/inmunología , Animales , Humanos , Glicoproteínas de Membrana/genética , Microglía/patología , Células Mieloides/patología , Receptores Inmunológicos/genética , Estados Unidos/epidemiología

RESUMEN

Genetic variants of TREM2, a protein expressed selectively by microglia in the brain, are associated with Alzheimer's disease (AD). Starting from an unbiased protein microarray screen, we identified a set of lipoprotein particles (including LDL) and apolipoproteins (including CLU/APOJ and APOE) as ligands of TREM2. Binding of these ligands by TREM2 was abolished or reduced by disease-associated mutations. Overexpression of wild-type TREM2 was sufficient to enhance uptake of LDL, CLU, and APOE in heterologous cells, whereas TREM2 disease variants were impaired in this activity. Trem2 knockout microglia showed reduced internalization of LDL and CLU. ß-amyloid (Aß) binds to lipoproteins and this complex is efficiently taken up by microglia in a TREM2-dependent fashion. Uptake of Aß-lipoprotein complexes was reduced in macrophages from human subjects carrying a TREM2 AD variant. These data link three genetic risk factors for AD and reveal a possible mechanism by which mutant TREM2 increases risk of AD. VIDEO ABSTRACT.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Apolipoproteínas/metabolismo , Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglía/metabolismo , Receptores Inmunológicos/metabolismo , Línea Celular , Humanos , Glicoproteínas de Membrana/genética , Unión Proteica , Receptores Inmunológicos/genética

RESUMEN

Recent reports suggest that botulinum neurotoxin (BoNT) A, which is widely used clinically to inhibit neurotransmission, can spread within networks of neurons to have distal effects, but this remains controversial. Moreover, it is not known whether other members of this toxin family are transferred between neurons. Here, we investigate the potential distal effects of BoNT/A, BoNT/D, and tetanus toxin (TeNT), using central neurons grown in microfluidic devices. Toxins acted upon the neurons that mediated initial entry, but all three toxins were also taken up, via an alternative pathway, into non-acidified organelles that mediated retrograde transport to the somato-dendritic compartment. Toxins were then released into the media, where they entered and exerted their effects upon upstream neurons. These findings directly demonstrate that these agents undergo transcytosis and interneuronal transfer in an active form, resulting in long-distance effects.

Asunto(s)

Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas/metabolismo , Hipocampo/metabolismo , Neuronas/metabolismo , Toxina Tetánica/metabolismo , Animales , Toxinas Botulínicas/toxicidad , Toxinas Botulínicas Tipo A/toxicidad , Comunicación Celular , Colorantes Fluorescentes/química , Hipocampo/citología , Hipocampo/efectos de los fármacos , Dispositivos Laboratorio en un Chip , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Cultivo Primario de Células , Transporte de Proteínas , Ratas , Toxina Tetánica/toxicidad

RESUMEN

Before exocytosis, vesicles must first become docked to the plasma membrane. The SNARE complex was originally hypothesized to mediate both the docking and fusion steps in the secretory pathway, but previous electron microscopy (EM) studies indicated that the vesicular SNARE protein synaptobrevin (syb) was dispensable for docking. In this paper, we studied the function of syb in the docking of large dense-core vesicles (LDCVs) in live PC12 cells using total internal reflection fluorescence microscopy. Cleavage of syb by a clostridial neurotoxin resulted in significant defects in vesicle docking in unfixed cells; these results were confirmed via EM using cells that were prepared using high-pressure freezing. The membrane-distal portion of its SNARE motif was critical for docking, whereas deletion of a membrane-proximal segment had little effect on docking but diminished fusion. Because docking was also inhibited by toxin-mediated cleavage of the target membrane SNAREs syntaxin and SNAP-25, syb might attach LDCVs to the plasma membrane through N-terminal assembly of trans-SNARE pairs.

Asunto(s)

Neuronas/metabolismo , Proteínas SNARE/química , Animales , Exocitosis , Procesamiento de Imagen Asistido por Computador/métodos , Inmunohistoquímica/métodos , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodos , Neurotoxinas/química , Células PC12 , Unión Proteica , Proteínas R-SNARE/química , Ratas , Vesículas Secretoras/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

Botulinum neurotoxin (BoNT) A and B are used to treat neuropathic disorders; if retargeted, these agents could be used to treat medical conditions that involve secretion from nonneuronal cells. Here, we report novel strategies for successfully retargeting BoNTs, and also tetanus neurotoxin (TeNT), to primary human blood monocyte-derived macrophages where BoNT/B inhibited the release of tumor necrosis factor-α, a cytokine that plays a key role in inflammation. Furthermore, mice treated with retargeted BoNT/B exhibited a significant reduction in macrophage (MΦ) recruitment, indicating that these toxins can be used to treat chronic inflammation.

Asunto(s)

Toxinas Botulínicas Tipo A/uso terapéutico , Neurotoxinas/uso terapéutico , Animales , Línea Celular , Enfermedad Crónica , Sistemas de Liberación de Medicamentos , Células HEK293 , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Neuronas/efectos de los fármacos , Neuronas/patología

RESUMEN

Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.

Asunto(s)

Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Toxina Tetánica/metabolismo , Animales , Biotinilación , Western Blotting , Células Cultivadas , Electrofisiología , Exocitosis/efectos de los fármacos , Femenino , Glicosilación/efectos de los fármacos , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Técnicas para Inmunoenzimas , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Ratas , Médula Espinal/efectos de los fármacos , Médula Espinal/embriología , Médula Espinal/metabolismo , Tasa de Supervivencia , Transmisión Sináptica , Proteína 2 de Membrana Asociada a Vesículas/metabolismo

RESUMEN

Styryl dyes (also referred to as FM dyes) become highly fluorescent upon binding to membranes and are often used to study synaptic vesicle recycling in neurons. To date, however, no direct comparisons of the fluorescent properties, or time-resolved (millisecond) measurements of dye-membrane binding and unbinding reactions, for all members of this family of probes have been reported. Here, we compare the fluorescence intensities of each member of the FM dye family when bound to membranes. This analysis included SGC5, a new lipophilic fluorescent dye with a unique structure. Fluorescence intensities depended on the length of the lipophilic tail of each dye, with a rank order as follows: SGC5 > FM1-84 > FM1-43 > SynaptoGreen C3 > FM2-10/FM4-64/FM5-95. Stopped-flow measurements revealed that dye hydrophobicity determined the affinity and departitioning rates for dye-membrane interactions. All of the dyes dissociated from membranes on the millisecond timescale, which is orders of magnitude faster than the overall destaining rate (timescale of seconds) of these dyes from presynaptic boutons. Departitioning kinetics were faster at higher temperatures, but were unaffected by pH or cholesterol. The data reported here aid interpretation of dye-release kinetics from single synaptic vesicles, and indicate that these probes dissociate from membranes on more rapid timescales than previously appreciated.

Asunto(s)

Membrana Celular/química , Colorantes Fluorescentes/química , Compuestos de Piridinio/química , Compuestos de Amonio Cuaternario/química , Animales , Colesterol/química , Fluorescencia , Hipocampo/química , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Liposomas/química , Neuronas/química , Terminales Presinápticos/química , Ratas , Temperatura