α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, includingParkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

SummaryThe aggresome is a key cytoplasmic organelle for sequestration and clearance of toxic protein aggregates. Although loading misfolded proteins cargos to dynein motors has been recognized as an important step in the aggresome formation process, the molecular machinery that mediates the association of cargos with the dynein motor is poorly understood. Here, we report a new aggresome-targeting pathway that involves isoforms of 14-3-3, a family of conserved regulatory proteins. 14-3-3 interacts with both the dynein-intermediate chain (DIC) and an Hsp70 co-chaperone Bcl-2-associated athanogene 3 (BAG3), thereby recruiting chaperone-associated protein cargos to dynein motors for their transport to aggresomes. This molecular cascade entails functional dimerization of 14-3-3, which we show to be crucial for the formation of aggresomes in both yeast and mammalian cells. These results suggest that 14-3-3 functions as a molecular adaptor to promote aggresomal targeting of misfolded protein aggregates and may link such complexes to inclusion bodies observed in various neurodegenerative diseases.

Ca 2؉/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the regulation of neuronal excitability in many systems. Recent studies suggest that local regulation of membrane potential can have important computational consequences for neuronal function. In Drosophila, CaMKII regulates the eag potassium channel, but if and how this regulation was spatially restricted was unknown. Calcium/calmodulin-dependent protein kinase II (CaMKII) 1 has been shown to regulate ion channels and neuronal excitability in both vertebrates (1-6) and invertebrates (4,5,(7)(8)(9)(10)(11). Regulation of excitability has been proposed as a mechanism by which neurons can globally modify their firing to keep spike rates in a scalable range in the presence of synapse-specific plasticity (12)(13)(14). Recently, it has become clear that regulation of excitability can also occur as a local phenomenon (15-19). Regional changes in excitability mediated by differing levels of A-type potassium currents were shown to be important for gating Hebbian plasticity in CA1 pyramidal cell dendrites and limiting propagation of action potentials (19). These effects have important implications for the computational abilities of neurons that integrate over many inputs. Both slow, expression level changes and fast, enzymatic modifications could underlie local phenomena, and in CA1, local regulation of excitability was associated with both regional differences in current density (19) and region-specific post-translational modifications of ion channels (18). Fast modulation offers the advantage of dynamic retooling of the computational capability of the neuron.For signal transduction pathways to effect fast local changes in excitability, the activity of effector enzymes has to be spatially restricted. The ability of protein kinases with many substrates to act with specificity to regulate cellular functions is increasingly being ascribed to interactions with scaffolding molecules that bring kinases and their substrates into close proximity. Signaling platforms range from small complexes containing only a few proteins to very complex cellular specializations like the postsynaptic density of the mammalian central nervous system (20). The advantages of scaffolding enzymes and substrates include enhancement of reaction rates because of elimination of diffusional barriers and increased local concentration of substrates and segregation of the enzyme to prevent reaction with other substrates. Localization of effector enzymes to sources of small molecule regulators can also establish very restricted signaling domains. Scaffolding may be particularly important for regulation of membrane-bound proteins in neurons; diffusion in the membrane bilayer is relatively slow, and the ability of soluble enzymes to access substrates can be influenced by the complex geometry of neurons.The regulation of firing in Drosophila motor axons involves the ether-à -go-go (eag) potassium channel. eag is the founding member of an evolutionarily conserved superfamily of voltageg...

Slob is a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium (K(Ca)) channel. A yeast two-hybrid screen with Slob as bait identifies the zeta isoform of 14-3-3 as a Slob-binding protein. Coimmunoprecipitation experiments from Drosophila heads and transfected cells confirm that 14-3-3 interacts with dSlo via Slob. All three proteins are colocalized presynaptically at Drosophila neuromuscular junctions. Two serine residues in Slob are required for 14-3-3 binding, and the binding is dynamically regulated in Drosophila by calcium/calmodulin-dependent kinase II (CaMKII) phosphorylation. 14-3-3 coexpression dramatically alters dSlo channel properties when wild-type Slob is present but not when a double serine mutant Slob that is incapable of binding 14-3-3 is present. The results provide evidence for a dSlo/Slob/14-3-3 regulatory protein complex.

Laughter is a nonverbal vocal expression that often communicates positive affect and cooperative intent in humans. Temporally coincident laughter occurring within groups is a potentially rich cue of affiliation to overhearers. We examined listeners’ judgments of affiliation based on brief, decontextualized instances of colaughter between either established friends or recently acquainted strangers. In a sample of 966 participants from 24 societies, people reliably distinguished friends from strangers with an accuracy of 53–67%. Acoustic analyses of the individual laughter segments revealed that, across cultures, listeners’ judgments were consistently predicted by voicing dynamics, suggesting perceptual sensitivity to emotionally triggered spontaneous production. Colaughter affords rapid and accurate appraisals of affiliation that transcend cultural and linguistic boundaries, and may constitute a universal means of signaling cooperative relationships.

Although it has been postulated that vesicle mobility is increased to enhance release of transmitters and neuropeptides, the mechanism responsible for increasing vesicle motion in nerve terminals and the effect of perturbing this mobilization on synaptic plasticity are unknown. Here, green fluorescent protein-tagged dense-core vesicles (DCVs) are imaged in Drosophila motor neuron terminals, where DCV mobility is increased for minutes after seconds of activity. Ca 2ϩ -induced Ca 2ϩ release from presynaptic endoplasmic reticulum (ER) is shown to be necessary and sufficient for sustained DCV mobilization. However, this ryanodine receptor (RyR)-mediated effect is short-lived and only initiates signaling. Calmodulin kinase II (CaMKII), which is not activated directly by external Ca 2ϩ influx, then acts as a downstream effector of released ER Ca 2ϩ . RyR and CaMKII are essential for post-tetanic potentiation of neuropeptide secretion. Therefore, the presynaptic signaling pathway for increasing DCV mobility is identified and shown to be required for synaptic plasticity.

Current brain spheroids or organoids derived from human induced pluripotent stem cells (hiPSCs) still lack a microglia component, the resident immune cells in the brain. The objective of this study is to engineer brain region-specific organoids from hiPSCs incorporated with isogenic microglia-like cells in order to enhance immune function. In this study, microglia-like cells were derived from hiPSCs using a simplified protocol with stage-wise growth factor induction, which expressed several phenotypic markers, including CD11b, IBA-1, CX3CR1, and P2RY12, and phagocytosed micron-size super-paramagnetic iron oxides. The derived cells were able to upregulate pro-inflammatory gene (TNF-α) and secrete anti-inflammatory cytokines (i.e., VEGF, TGF-β1, and PGE2) when stimulated with amyloid β42 oligomers, lipopolysaccharides, or dexamethasone. The derived isogenic dorsal cortical (higher expression of TBR1 and PAX6) and ventral (higher expression of NKX2.1 and PROX1) spheroids/organoids displayed action potentials and synaptic activities. Co-culturing the microglia-like cells (MG) with the dorsal (D) or ventral (V) organoids showed differential migration ability, intracellular Ca 2+ signaling, and the response to pro-inflammatory stimuli (V-MG group had higher TNF-α and TREM2 expression). Transcriptome analysis exhibited 37 microglia-related genes that were differentially expressed in MG and D-MG groups. In addition, the hybrid D-MG spheroids exhibited higher levels of immunoreceptor genes in activating members, but the MG group contained higher levels for most of genes in inhibitory members (except SIGLEC5 and CD200). This study should advance our understanding of the microglia function in brain-like tissue and establish a transformative approach to modulate cellular microenvironment toward the goal of treating various neurological disorders.

14-3-3 is a family of regulatory proteins highly expressed in the brain. Previous invertebrate studies have demonstrated the importance of 14-3-3 in the regulation of synaptic functions and learning and memory. However, the in vivo role of 14-3-3 in these processes has not been determined using mammalian animal models. Here, we report the behavioral and electrophysiological characterization of a new animal model of 14-3-3 proteins. These transgenic mice, considered to be a 14-3-3 functional knock-out, express a known 14-3-3 inhibitor in various brain regions of differentfounderlines.Weidentifyafounder-specificimpairmentinhippocampal-dependentlearningandmemorytasks,aswellasacorrelated suppression in long-term synaptic plasticity of the hippocampal synapses. Moreover, hippocampal synaptic NMDA receptor levels are selectively reduced in the transgenic founder line that exhibits both behavioral and synaptic plasticity deficits. Collectively, our findings provide evidence that 14-3-3 is a positive regulator of associative learning and memory at both the behavioral and cellular level.