Neuronal Glutamate Transporters Control Dopaminergic Signaling and Compulsive Behaviors.

There is an ongoing debate on the contribution of the neuronal glutamate transporter EAAC1 to the onset of compulsive behaviors. Here, we used behavioral, electrophysiological, molecular, and viral approaches in male and female mice to identify the molecular and cellular mechanisms by which EAAC1 controls the execution of repeated motor behaviors. Our findings show that, in the striatum, a brain region implicated with movement execution, EAAC1 limits group I metabotropic glutamate receptor (mGluRI) activation, facilitates D1 dopamine receptor (D1R) expression, and ensures long-term synaptic plasticity. Blocking mGluRI in slices from mice lacking EAAC1 restores D1R expression and synaptic plasticity. Conversely, activation of intracellular signaling pathways coupled to mGluRI in D1R-containing striatal neurons of mice expressing EAAC1 leads to reduced D1R protein level and increased stereotyped movement execution. These findings identify new molecular mechanisms by which EAAC1 can shape glutamatergic and dopaminergic signals and control repeated movement execution.SIGNIFICANCE STATEMENT Genetic studies implicate Slc1a1, a gene encoding the neuronal glutamate transporter EAAC1, with obsessive-compulsive disorder (OCD). EAAC1 is abundantly expressed in the striatum, a brain region that is hyperactive in OCD. What remains unknown is how EAAC1 shapes synaptic function in the striatum. Our findings show that EAAC1 limits activation of metabotropic glutamate receptors (mGluRIs) in the striatum and, by doing so, promotes D1 dopamine receptor (D1R) expression. Targeted activation of signaling cascades coupled to mGluRIs in mice expressing EAAC1 reduces D1R expression and triggers repeated motor behaviors. These findings provide new information on the molecular basis of OCD and suggest new avenues for its treatment.

ENGINEERED NANOBODIES WITH PROGRAMMABLE TARGET ANTIGEN PROTEOLYSIS (PTAP) FUSIONS REGULATE INTRACELLULAR ALPHA-SYNUCLEIN IN VITRO AND IN VIVO.

Alpha-synuclein (αSyn) aggregation and the formation of Lewy pathology (LP) is a foundational pathophysiological phenomenon in synucleinopathies. Delivering therapeutic single-chain and single-domain antibodies that bind pathogenic targets can disrupt intracellular aggregation. The fusion of antibody fragments to a negatively-charged proteasomal targeting motif (PEST) creates bifunctional constructs that enhance both solubility and turnover. With sequence-specific point mutations of PEST sequences that modulate proteasomal degradation efficiency, we report the creation of Programmable Target Antigen Proteolysis (PTAP) technology that can provide graded control over the levels of target antigens. We have previously demonstrated our lead anti-αSyn intrabody, VH14-PEST, is capable of reducing the pathological burden of synucleinopathy in vitro and in vivo. Here, we report a family of fully humanized VH14-PTAP constructs for controllable, therapeutic targeting of intracellular α-Syn. In cells, we demonstrate successful target engagement and efficacy of VH14-hPEST intrabodies, and validate proof-of-principle in human cells using 3D human organoids derived from PD-patient induced pluripotent stem cells (iPSC). In two synuclein-based rat models, PTAP intrabodies attenuated nigral αSyn pathology, preserved nigrostriatal dopaminergic tone, and slowed the propagation of αSyn pathology. These data demonstrate the potency of intracellular αSyn targeting as a method to alleviate pathology and highlight the potential clinical utility of PTAP intrabodies.

Fully Human Bifunctional Intrabodies Achieve Graded Reduction of Intracellular Tau and Rescue Survival of MAPT Mutation iPSC-derived Neurons.

Tau protein aggregation is a hallmark of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP), spurring development of tau-lowering therapeutic strategies. Here, we report fully human bifunctional anti-tau-PEST intrabodies that bind the mid-domain of tau to block aggregation and degrade tau via the proteasome using the ornithine decarboxylase (ODC) PEST degron. They effectively reduced tau protein in human iPSC-derived cortical neurons in 2D cultures and 3D organoids, including those with the disease-associated tau mutations R5L, N279K, R406W, and V337M. Anti-tau-hPEST intrabodies facilitated efficient ubiquitin-independent proteolysis, in contrast to tau-lowering approaches that rely on the cell's ubiquitination system. Importantly, they counteracted the proteasome impairment observed in V337M patient-derived cortical neurons and significantly improved neuronal survival. By serial mutagenesis, we created variants of the PEST degron that achieved graded levels of tau reduction. Moderate reduction was as effective as high reduction against tau V337M-induced neural cell death.

Neuronal glutamate transporters control reciprocal inhibition and gain modulation in D1 medium spiny neurons.

Understanding the function of glutamate transporters has broad implications for explaining how neurons integrate information and relay it through complex neuronal circuits. Most of what is currently known about glutamate transporters, specifically their ability to maintain glutamate homeostasis and limit glutamate diffusion away from the synaptic cleft, is based on studies of glial glutamate transporters. By contrast, little is known about the functional implications of neuronal glutamate transporters. The neuronal glutamate transporter EAAC1 is widely expressed throughout the brain, particularly in the striatum, the primary input nucleus of the basal ganglia, a region implicated with movement execution and reward. Here, we show that EAAC1 limits synaptic excitation onto a population of striatal medium spiny neurons identified for their expression of D1 dopamine receptors (D1-MSNs). In these cells, EAAC1 also contributes to strengthen lateral inhibition from other D1-MSNs. Together, these effects contribute to reduce the gain of the input-output relationship and increase the offset at increasing levels of synaptic inhibition in D1-MSNs. By reducing the sensitivity and dynamic range of action potential firing in D1-MSNs, EAAC1 limits the propensity of mice to rapidly switch between behaviors associated with different reward probabilities. Together, these findings shed light on some important molecular and cellular mechanisms implicated with behavior flexibility in mice.

Methylene blue is a potent and broad-spectrum inhibitor against Zika virus in vitro and in vivo.

Many flaviviruses including the Dengue virus (DENV), Zika virus (ZIKV), West Nile virus, Yellow Fever virus, and Japanese encephalitis virus are significant human pathogens, unfortunately without any specific therapy. Here, we demonstrate that methylene blue, an FDA-approved drug, is a broad-spectrum and potent antiviral against Zika virus and Dengue virus both in vitro and in vivo. We found that methylene blue can considerably inhibit the interactions between viral protease NS3 and its NS2B co-factor, inhibit viral protease activity, inhibit viral growth, protect 3D mini-brain organoids from ZIKV infection, and reduce viremia in a mouse model. Mechanistic studies confirmed that methylene blue works in both entry and post entry steps, reduces virus production in replicon cells and inhibited production of processed NS3 protein. Overall, we have shown that methylene blue is a potent antiviral for management of flavivirus infections, particularly for Zika virus. As an FDA-approved drug, methylene blue is well-tolerated for human use. Therefore, methylene blue represents a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.

JMX0207, a Niclosamide Derivative with Improved Pharmacokinetics, Suppresses Zika Virus Infection Both In Vitro and In Vivo.

Flaviviruses causes significant human disease. Recent outbreaks of the Zika virus highlight the need to develop effective therapies for this class of viruses. Previously we identified niclosamide as a broad-spectrum inhibitor for flaviviruses by targeting the interface between viral protease NS3 and its cofactor NS2B. Here, we screened a small library of niclosamide derivatives and identified a new analogue with improved pharmacokinetic properties. Compound JMX0207 showed improved efficacy in inhibition of the molecular interaction between NS3 and NS2B, better inhibition of viral protease function, and enhanced antiviral efficacy in the cell-based antiviral assay. The derivative also significantly reduced Zika virus infection on 3D mini-brain organoids derived from pluripotent neural stem cells. Intriguingly, the compound significantly reduced viremia in a Zika virus (ZIKV) animal model. In summary, a niclosamide derivative, JMX0207, was identified, which shows improved pharmacokinetics and efficacy against Zika virus both in vitro and in vivo.

Circadian Modulation of Neurons and Astrocytes Controls Synaptic Plasticity in Hippocampal Area CA1.

Most animal species operate according to a 24-h period set by the suprachiasmatic nucleus (SCN) of the hypothalamus. The rhythmic activity of the SCN modulates hippocampal-dependent memory, but the molecular and cellular mechanisms that account for this effect remain largely unknown. Here, we identify cell-type-specific structural and functional changes that occur with circadian rhythmicity in neurons and astrocytes in hippocampal area CA1. Pyramidal neurons change the surface expression of NMDA receptors. Astrocytes change their proximity to synapses. Together, these phenomena alter glutamate clearance, receptor activation, and integration of temporally clustered excitatory synaptic inputs, ultimately shaping hippocampal-dependent learning in vivo. We identify corticosterone as a key contributor to changes in synaptic strength. These findings highlight important mechanisms through which neurons and astrocytes modify the molecular composition and structure of the synaptic environment, contribute to the local storage of information in the hippocampus, and alter the temporal dynamics of cognitive processing.

Methamphetamine-induced apoptosis in glial cells examined under marker-free imaging modalities.

We used phase microscopy and Raman spectroscopic measurements to assess the response of in vitro rat C6 glial cells following methamphetamine treatment in real time. Digital holographic microscopy (DHM) and three-dimensional (3-D) tomographic nanoscopy allow measurements of live cell cultures, which yield information about cell volume changes. Tomographic phase imaging provides 3-D information about the refractive index distribution associated with the morphology of biological samples. DHM provides similar information, but for a larger population of cells. Morphological changes in cells are associated with alterations in cell cycle and initiation of cell death mechanisms. Raman spectroscopy measurements provide information about chemical changes within the cells. Our Raman data indicate that the chemical changes in proteins preceded morphological changes, which were seen with DHM. Our study also emphasizes that tomographic phase imaging, DHM, and Raman spectroscopy are imaging tools that can be utilized for noninvasive simultaneous monitoring of morphological and chemical changes in cells during apoptosis and can also be used to monitor other dynamic cell processes.