Dietary Zinc Supplementation Prevents Autism Related Behaviors and Striatal Synaptic Dysfunction in Shank3 Exon 13-16 Mutant Mice.

The SHANK family of synaptic proteins (SHANK1-3) are master regulators of the organizational structure of excitatory synapses in the brain. Mutations in SHANK1-3 are prevalent in patients with autism spectrum disorders (ASD), and loss of one copy of SHANK3 causes Phelan-McDermid Syndrome, a syndrome in which Autism occurs in >80% of cases. The synaptic stability of SHANK3 is highly regulated by zinc, driving the formation of postsynaptic protein complexes and increases in excitatory synaptic strength. As ASD-associated SHANK3 mutations retain responsiveness to zinc, here we investigated how increasing levels of dietary zinc could alter behavioral and synaptic deficits that occur with ASD. We performed behavioral testing together with cortico-striatal slice electrophysiology on a Shank3 -/- mouse model of ASD (Shank3 ex13-1616-/-), which displays ASD-related behaviors and structural and functional deficits at striatal synapses. We observed that 6 weeks of dietary zinc supplementation in Shank3 ex13-16-/- mice prevented ASD-related repetitive and anxiety behaviors and deficits in social novelty recognition. Dietary zinc supplementation also increased the recruitment of zinc sensitive SHANK2 to synapses, reduced synaptic transmission specifically through N-methyl-D-aspartate (NMDA)-type glutamate receptors, reversed the slowed decay tau of NMDA receptor (NMDAR)-mediated currents and occluded long term potentiation (LTP) at cortico-striatal synapses. These data suggest that alterations in NMDAR function underlie the lack of NMDAR-dependent cortico-striatal LTP and contribute to the reversal of ASD-related behaviors such as compulsive grooming. Our data reveal that dietary zinc alters neurological function from synapses to behavior, and identifies dietary zinc as a potential therapeutic agent in ASD.

Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets.

Interest in the human microbiome has increased dramatically in the last decade. However, much of this research has focused on bacteria, while the composition and roles of their fungal counterparts remain less understood. Furthermore, a variety of methodological approaches have been applied, and the comparability between studies is unclear. This study compared four primer pairs targeting the small subunit (SSU) rRNA (18S), ITS1, ITS2, and large subunit (LSU) rRNA (26S) genomic regions for their ability to accurately characterize fungal communities typical of the human mycobiota. All four target regions of 21 individual fungal mock community taxa were capable of being amplified adequately and sequenced. Mixed mock community analyses revealed marked variability in the ability of each primer pair to accurately characterize a complex community. ITS target regions outperformed LSU and SSU. Of the ITS regions, ITS1 failed to generate sequences for Yarrowia lipolytica and all three Malassezia species when in a mixed community. These findings were further supported in studies of human sinonasal and mouse fecal samples. Based on these analyses, previous studies using ITS1, SSU, or LSU markers may omit key taxa that are identified by the ITS2 marker. Of methods commonly used in human mycobiota studies to date, we recommend selection of the ITS2 marker. Further investigation of more recently developed fungal primer options will be essential to ultimately determine the optimal methodological approach by which future human mycobiota studies ought to be standardized.

SAP97-mediated rescue of NMDA receptor surface distribution in a neuronal model of Huntington's disease.

Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expansion of the CAG repeat tract in the HTT gene, leading to motor, cognitive, and psychiatric symptoms. At the cellular level, NMDA-type glutamate receptors are upregulated at glutamatergic extrasynaptic sites in HD, triggering cell death signaling pathways and driving HD neurodegeneration. Extrasynaptic and synaptic glutamate receptor trafficking and surface distribution are regulated by the α and ß N-terminal isoforms of SAP97, a postsynaptic density protein localized at glutamatergic synapses. Here we examined the surface distribution of NMDARs and AMPARs in a cellular model of HD, and whether the manipulation of individual SAP97 isoforms can regulate receptor distribution in diseased neurons. Using dSTORM super-resolution imaging, we reveal that mutant HTT drives the elevation of extrasynaptic NMDAR clusters located 100-500 nm from the postsynaptic density. This was accompanied by a decline in synaptic NMDAR-mediated currents while surface NMDAR-mediated currents remained unchanged. These effects were induced within 3 days of mutant HTT expression in rat hippocampal neurons in vitro, and were specific for NMDARs and not observed with AMPARs. Intriguingly, upregulation of either α- or ßSAP97 expression increased synaptic and/or perisynaptic NMDAR localization and prevented the shift of NMDARs to extrasynaptic sites in mutant HTT neurons. This was accompanied by the rescue of normal synaptic NMDAR-mediated currents. Taken together, our high-resolution data reveals plasticity in surface NMDAR localization driven by mutant HTT and identifies the similar but independent roles of SAP97 N-terminal isoforms in maintaining normal synaptic function in pathological states.

α-synuclein transfer through tunneling nanotubes occurs in SH-SY5Y cells and primary brain pericytes from Parkinson's disease patients.

Parkinson's disease (PD) is characterized by the presence of inclusions known as Lewy bodies, which mainly consist of α-synuclein (α-syn) aggregates. There is growing evidence that α-syn self-propagates in non-neuronal cells, thereby contributing to the progression and spread of PD pathology in the brain. Tunneling nanotubes (TNTs) are long, thin, F-actin-based membranous channels that connect cells and have been proposed to act as conduits for α-syn transfer between cells. SH-SY5Y cells and primary human brain pericytes, derived from postmortem PD brains, frequently form TNTs that allow α-syn transfer and long-distance electrical coupling between cells. Pericytes in situ contain α-syn precipitates like those seen in neurons. Exchange through TNTs was rapid, but dependent on the size of the protein. Proteins were able to spread throughout a network of cells connected by TNTs. Transfer through TNTs was not restricted to α-syn; fluorescent control proteins and labeled membrane were also exchanged through TNTs. Most importantly the formation of TNTs and transfer continued during mitosis. Together, our results provide a detailed description of TNTs in SH-SY5Y cells and human brain PD pericytes, demonstrating their role in α-syn transfer and further emphasize the importance that non-neuronal cells, such as pericytes play in disease progression.

AMPA Receptors as Therapeutic Targets for Neurological Disorders.

Almost every neurological disease directly or indirectly affects synapse function in the brain. However, these diseases alter synapses through different mechanisms, ultimately resulting in altered synaptic transmission and/or plasticity. Glutamate is the major neurotransmitter that mediates excitatory synaptic transmission in the brain through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. These receptors have therefore been identified as a target for the development of therapeutic treatments for neurological disorders including epilepsy, neurodegenerative diseases, autism, and drug addiction. The fact that AMPA receptors play a dominant role throughout the brain raises the significant challenge of selectively targeting only those regions affected by disease, and clinical trials have raised doubt regarding the feasibility of specifically targeting AMPA receptors for new therapeutic options. Benzamide compounds that act as positive allosteric AMPA receptor modulators, known as AMPAkines, can act on specific brain regions and were initially proposed to revolutionize the treatment of cognitive deficits associated with neurological disorders. Their therapeutic potential has since declined due to inconsistent results in clinical trials. However, recent advances in basic biomedical research are significantly increasing our knowledge of AMPA receptor structure, binding sites, and interactions with auxiliary proteins. In particular, the large complex of postsynaptic proteins that interact with AMPA receptor subunits have been shown to control AMPA receptor insertion, location, pharmacology, synaptic transmission, and plasticity. These proteins are now being considered as alternative therapeutic target sites for modulating AMPA receptors in neurological disorders.

Paired whole cell recordings in organotypic hippocampal slices.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

The anchoring protein SAP97 influences the trafficking and localisation of multiple membrane channels.

SAP97 is a member of the MAGUK family of proteins that play a major role in the trafficking and targeting of membrane ion channels and cytosolic structural proteins in multiple cell types. Within neurons, SAP97 is localised throughout the secretory trafficking pathway and at the postsynaptic density (PSD). SAP97 differs from other MAGUK family members largely in its long N-terminus and in the sequences between the SH3 and GUK domains, where SAP97 undergoes significant alternative splicing to produce multiple SAP97 isoforms. These splice insertions endow SAP97 with differential cellular localisation patterns and functional roles within neurons. With regard to membrane ion channels, SAP97 forms multi-protein complexes with AMPA and NMDA-type glutamate receptors, and Kv1.4, Kv4.2, and Kir2.2 potassium channels, playing a major role in trafficking and anchoring ion channel surface expression. This highlights SAP97 not only as a regulator of neuronal excitability, synaptic function and plasticity in the brain, but also as a target for the pathophysiology of a number of neurological disorders. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.