Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways.

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.

Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation.

Length-dependent axonopathy of the corticospinal tract causes lower limb spasticity and is characteristic of several neurological disorders, including hereditary spastic paraplegia (HSP) and amyotrophic lateral sclerosis. Mutations in Trk-fused gene (TFG) have been implicated in both diseases, but the pathomechanisms by which these alterations cause neuropathy remain unclear. Here, we biochemically and genetically define the impact of a mutation within the TFG coiled-coil domain, which underlies early-onset forms of HSP. We find that the TFG (p.R106C) mutation alters compaction of TFG ring complexes, which play a critical role in the export of cargoes from the endoplasmic reticulum (ER). Using CRISPR-mediated genome editing, we engineered human stem cells that express the mutant form of TFG at endogenous levels and identified specific defects in secretion from the ER and axon fasciculation following neuronal differentiation. Together, our data highlight a key role for TFG-mediated protein transport in the pathogenesis of HSP.

Doc2-mediated superpriming supports synaptic augmentation.

Various forms of synaptic plasticity underlie aspects of learning and memory. Synaptic augmentation is a form of short-term plasticity characterized by synaptic enhancement that persists for seconds following specific patterns of stimulation. The mechanisms underlying this form of plasticity are unclear but are thought to involve residual presynaptic Ca2+ Here, we report that augmentation was reduced in cultured mouse hippocampal neurons lacking the Ca2+ sensor, Doc2; other forms of short-term enhancement were unaffected. Doc2 binds Ca2+ and munc13 and translocates to the plasma membrane to drive augmentation. The underlying mechanism was not associated with changes in readily releasable pool size or Ca2+ dynamics, but rather resulted from superpriming a subset of synaptic vesicles. Hence, Doc2 forms part of the Ca2+-sensing apparatus for synaptic augmentation via a mechanism that is molecularly distinct from other forms of short-term plasticity.

An Engineered Metal Sensor Tunes the Kinetics of Synaptic Transmission.

The Ca(2+) sensor synaptotagmin-1 (syt-1) regulates neurotransmitter release by interacting with anionic phospholipids. Here we test the idea that the intrinsic kinetics of syt-membrane interactions determine, in part, the time course of synaptic transmission. To tune the kinetics of this interaction, we grafted structural elements from the slowest isoform, syt-7, onto the fastest isoform, syt-1, resulting in a chimera with intermediate kinetic properties. Moreover, the chimera coupled a physiologically irrelevant metal, Sr(2+), to membrane fusion in vitro. When substituted for syt-1 in mouse hippocampal neurons, the chimera slowed the kinetics of synaptic transmission. Neurons expressing the chimera also evinced rapid and efficient Sr(2+) triggered release, in contrast to the weak response of neurons expressing syt-1. These findings reveal presynaptic sensor-membrane interactions as a major factor regulating the speed of the release machinery. Finally, the chimera failed to clamp the elevated spontaneous fusion rate exhibited by syt-1 KO neurons, indicating that the metal binding loops of syt-1 regulate the two modes of release by distinct mechanisms. SIGNIFICANCE STATEMENT: In calcium, synaptotagmin-1 triggers neurotransmitter release by interacting with membranes. Here, we demonstrate that intrinsic properties of this interaction control the time course of synaptic transmission. We engineered a "chimera" using synaptotagmin-1 and elements of a slower isoform, synaptotagmin-7. When expressed in neurons, the chimera slowed the rate of neurotransmitter release. Furthermore, unlike native synaptotagmin-1, the chimera was able to function robustly in the presence of strontium-a metal not present in cells. We exploited this ability to show that a key function of synaptotagmin-1 is to penetrate cell membranes. This work sheds light on fundamental mechanisms of neurotransmitter release.

Deterministic HOX patterning in human pluripotent stem cell-derived neuroectoderm.

Colinear HOX expression during hindbrain and spinal cord development diversifies and assigns regional neural phenotypes to discrete rhombomeric and vertebral domains. Despite the precision of HOX patterning in vivo, in vitro approaches for differentiating human pluripotent stem cells (hPSCs) to posterior neural fates coarsely pattern HOX expression thereby generating cultures broadly specified to hindbrain or spinal cord regions. Here, we demonstrate that successive activation of fibroblast growth factor, Wnt/ß-catenin, and growth differentiation factor signaling during hPSC differentiation generates stable, homogenous SOX2(+)/Brachyury(+) neuromesoderm that exhibits progressive, full colinear HOX activation over 7 days. Switching to retinoic acid treatment at any point during this process halts colinear HOX activation and transitions the neuromesoderm into SOX2(+)/PAX6(+) neuroectoderm with predictable, discrete HOX gene/protein profiles that can be further differentiated into region-specific cells, e.g., motor neurons. This fully defined approach significantly expands capabilities to derive regional neural phenotypes from diverse hindbrain and spinal cord domains.

Diffusion tensor imaging of white matter networks in individuals with current and remitted alcohol use disorders and comorbid conditions.

Individuals with alcohol use disorders show white matter abnormality relative to normal samples, but differences in white matter profiles have not yet been investigated as a function of abstinence. Individuals with current alcohol use disorders (AUD-C; n = 10), individuals with alcohol use disorders in remission for at least 1 year (AUD-R; n = 9), and healthy control participants (HC; n = 15) matched to alcohol groups on age and smoking status underwent MRI. Diffusion tensor imaging (DTI) data were analyzed using tract-based spatial statistics (TBSS). Compared with HC, AUD-C showed reduced axial diffusivity in bilateral frontal and temporal white matter. In AUD-R, lower fractional anisotropy relative to HC was widespread in bilateral parietal regions. A combined AUD-C and AUD-R group had decreased fractional anisotropy primarily in the fornix and thalamus. In conclusion, AUD-R manifested damage in parietal regions integral to processing of visuospatial information and self-awareness whereas AUD-C showed abnormal diffusivity in fronto-temporal regions that regulate impulsivity, attention, and memory. As a combined group, AUD individuals exhibited abnormality in subcortical areas associated with sensory processing and memory. White matter differences in individuals with AUD may be attributable to premorbid vulnerability or persisting effects of alcohol abuse, but the pattern of abnormality across groups suggests that these abnormalities may be secondary to alcohol use.

Evoked and intrinsic asymmetries during auditory attention: implications for the contralateral and neglect models of functioning.

Unlike the visual system, a direct mapping of extrapersonal space does not exist within human auditory cortex (AC). Thus, models (contralateral bias vs. neglect) of how auditory spatial attention is allocated remain debated, as does the role of hemispheric asymmetries. To further examine these questions, 27 participants completed an exogenous auditory orienting task while undergoing functional magnetic resonance imaging. Resting-state data were also collected to characterize intrinsic activity within the AC. Current results provide the first evidence of hemispheric specialization in the "where" (right secondary AC) auditory processing stream during both evoked (orienting task) and intrinsic (resting-state data) activity, suggesting that spontaneous and evoked activity may be synchronized by similar cortical hierarchies. Strong evidence for a contralateral bias model was observed during rapid deployment stages (facilitation) of auditory attention in bilateral AC. However, contralateral bias increased for left and decreased for right AC (neglect model) after longer stimulus onset asynchronies (inhibition of return), suggesting a role for higher-order cortical structures in modulating AC functioning. Prime candidates for attentional modulation include the frontoparietal network, which demonstrated right hemisphere lateralization across multiple attentional states.

Adolescent substance abuse: the effects of alcohol and marijuana on neuropsychological performance.

BACKGROUND: Adolescence is a period in which cognition and brain undergo dramatic parallel development. Whereas chronic use of alcohol and marijuana is known to cause cognitive impairments in adults, far less is known about the effect of these substances of abuse on adolescent cognition, including possible interactions with developmental processes. METHODS: Neuropsychological performance, alcohol use, and marijuana use were assessed in 48 adolescents (ages 12 to 18), recruited in 3 groups: a healthy control group (HC, n = 15), a group diagnosed with substance abuse or dependence (SUD, n = 19), and a group with a family history positive for alcohol use disorder (AUD) but no personal substance use disorder (FHP, n = 14). Age, drinks per drinking day (DPDD), percentage days drinking, and percentage days using marijuana were considered as covariates in a MANCOVA in which 6 neuropsychological composites (Verbal Reasoning, Visuospatial Ability, Executive Function, Memory, Attention, and Processing Speed) served as dependent variables. RESULTS: More DPDD predicted poorer performance on Attention and Executive Function composites, and more frequent use of marijuana was associated with poorer Memory performance. In separate analyses, adolescents in the SUD group had lower scores on Attention, Memory, and Processing Speed composites, and FHP adolescents had poorer Visuospatial Ability. CONCLUSIONS: In combination, these analyses suggest that heavy alcohol use in adolescence leads to reduction in attention and executive functioning and that marijuana use exerts an independent deleterious effect on memory. At the same time, premorbid deficits associated with family history of AUD appeared to be specific to visuospatial ability.