Synthetic Polymers Provide a Robust Substrate for Functional Neuron Culture.

Substrates for neuron culture and implantation are required to be both biocompatible and display surface compositions that support cell attachment, growth, differentiation, and neural activity. Laminin, a naturally occurring extracellular matrix protein is the most widely used substrate for neuron culture and fulfills some of these requirements, however, it is expensive, unstable (compared to synthetic materials), and prone to batch-to-batch variation. This study uses a high-throughput polymer screening approach to identify synthetic polymers that supports the in vitro culture of primary mouse cerebellar neurons. This allows the identification of materials that enable primary cell attachment with high viability even under "serum-free" conditions, with materials that support both primary cells and neural progenitor cell attachment with high levels of neuronal biomarker expression, while promoting progenitor cell maturation to neurons.

Candidate CSPG4 mutations and induced pluripotent stem cell modeling implicate oligodendrocyte progenitor cell dysfunction in familial schizophrenia.

Schizophrenia is highly heritable, yet its underlying pathophysiology remains largely unknown. Among the most well-replicated findings in neurobiological studies of schizophrenia are deficits in myelination and white matter integrity; however, direct etiological genetic and cellular evidence has thus far been lacking. Here, we implement a family-based approach for genetic discovery in schizophrenia combined with functional analysis using induced pluripotent stem cells (iPSCs). We observed familial segregation of two rare missense mutations in Chondroitin Sulfate Proteoglycan 4 (CSPG4) (c.391G > A [p.A131T], MAF 7.79 × 10-5 and c.2702T > G [p.V901G], MAF 2.51 × 10-3). The CSPG4A131T mutation was absent from the Swedish Schizophrenia Exome Sequencing Study (2536 cases, 2543 controls), while the CSPG4V901G mutation was nominally enriched in cases (11 cases vs. 3 controls, P = 0.026, OR 3.77, 95% CI 1.05-13.52). CSPG4/NG2 is a hallmark protein of oligodendrocyte progenitor cells (OPCs). iPSC-derived OPCs from CSPG4A131T mutation carriers exhibited abnormal post-translational processing (P = 0.029), subcellular localization of mutant NG2 (P = 0.007), as well as aberrant cellular morphology (P = 3.0 × 10-8), viability (P = 8.9 × 10-7), and myelination potential (P = 0.038). Moreover, transfection of healthy non-carrier sibling OPCs confirmed a pathogenic effect on cell survival of both the CSPG4A131T (P = 0.006) and CSPG4V901G (P = 3.4 × 10-4) mutations. Finally, in vivo diffusion tensor imaging of CSPG4A131T mutation carriers demonstrated a reduction of brain white matter integrity compared to unaffected sibling and matched general population controls (P = 2.2 × 10-5). Together, our findings provide a convergence of genetic and functional evidence to implicate OPC dysfunction as a candidate pathophysiological mechanism of familial schizophrenia.

Activity-Dependent Myelination of Parvalbumin Interneurons Mediated by Axonal Morphological Plasticity.

Axonal myelination of neocortical pyramidal neurons is modulated dynamically by neuronal activity. Recent studies have shown that a substantial proportion of neocortical myelin content is contributed by fast-spiking, parvalbumin (PV)-positive interneurons. However, it remains unknown whether the myelination of PV+ interneurons is also modulated by intrinsic activity. Here, we used cell-type-specific Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) in adult mice to activate a sparse population of medial prefrontal cortex (mPFC) PV+ interneurons. Using single-cell axonal reconstructions, we found that DREADD-stimulated PV+ interneurons exhibited a nearly two-fold increase in total length of myelination, predominantly mediated by a parallel increase of axonal arborization and number of internodes. In contrast, the distribution of axonal interbranch segment distance and myelin internode length were not altered significantly. Topographical analysis revealed that myelination of DREADD-stimulated cells extended to higher axonal branch orders while retaining a similar interbranch distance threshold for myelination. Together, our results demonstrate that chemogenetically induced neuronal activity increases the myelination of neocortical PV+ interneurons mediated at least in part by an elaboration of their axonal morphology.SIGNIFICANCE STATEMENT Myelination is the wrapping of an axon to optimize conduction velocity in an energy-efficient manner. Previous studies have shown that myelination of neocortical pyramidal neurons is experience and activity dependent. We now show that activity-dependent myelin plasticity in the adult neocortex extends to parvalbumin (PV)-expressing fast-spiking interneurons. Chemogenetic stimulation of PV interneurons in the medial prefrontal cortex (mPFC) significantly enhanced axonal myelination, which was paralleled by an increase in axonal arborization. This suggests that activity-dependent axonal plasticity may involve changes in both structural morphology and myelination. Such multicomponent plasticity reveals an unexpected repertoire of anatomical parameters available for optimizing and adapting neuronal networks in response to experience.

An expandable embryonic stem cell-derived Purkinje neuron progenitor population that exhibits in vivo maturation in the adult mouse cerebellum.

The directed differentiation of patient-derived induced pluripotent stem cells into cell-type specific neurons has inspired the development of therapeutic discovery for neurodegenerative diseases. Many forms of ataxia result from degeneration of cerebellar Purkinje cells, but thus far it has not been possible to efficiently generate Purkinje neuron (PN) progenitors from human or mouse pluripotent stem cells, let alone to develop a methodology for in vivo transplantation in the adult cerebellum. Here, we present a protocol to obtain an expandable population of cerebellar neuron progenitors from mouse embryonic stem cells. Our protocol is characterized by applying factors that promote proliferation of cerebellar progenitors. Cerebellar progenitors isolated in culture from cell aggregates contained a stable subpopulation of PN progenitors that could be expanded for up to 6 passages. When transplanted into the adult cerebellum of either wild-type mice or a strain lacking Purkinje cells (L7cre-ERCC1 knockout), GFP-labeled progenitors differentiated in vivo to establish a population of calbindin-positive cells in the molecular layer with dendritic trees typical of mature PNs. We conclude that this protocol may be useful for the generation and maturation of PNs, highlighting the potential for development of a regenerative medicine approach to the treatment of cerebellar neurodegenerative diseases.

Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10-2474.

A recent phase 1 trial of the fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474 led to the death of one volunteer and produced mild-to-severe neurological symptoms in four others. Although the cause of the clinical neurotoxicity is unknown, it has been postulated, given the clinical safety profile of other tested FAAH inhibitors, that off-target activities of BIA 10-2474 may have played a role. Here we use activity-based proteomic methods to determine the protein interaction landscape of BIA 10-2474 in human cells and tissues. This analysis revealed that the drug inhibits several lipases that are not targeted by PF04457845, a highly selective and clinically tested FAAH inhibitor. BIA 10-2474, but not PF04457845, produced substantial alterations in lipid networks in human cortical neurons, suggesting that promiscuous lipase inhibitors have the potential to cause metabolic dysregulation in the nervous system.

Transient and sustained afterdepolarizations in accessory olfactory bulb mitral cells are mediated by distinct mechanisms that are differentially regulated by neuromodulators.

Social interactions between mammalian conspecifics rely heavily on molecular communication via the main and accessory olfactory systems. These two chemosensory systems show high similarity in the organization of information flow along their early stages: social chemical cues are detected by the sensory neurons of the main olfactory epithelium and the vomeronasal organ. These neurons then convey sensory information to the main (MOB) and accessory (AOB) olfactory bulbs, respectively, where they synapse upon mitral cells that project to higher brain areas. Yet, the functional difference between these two chemosensory systems remains unclear. We have previously shown that MOB and AOB mitral cells exhibit very distinct intrinsic biophysical properties leading to different types of information processing. Specifically, we found that unlike MOB mitral cells, AOB neurons display persistent firing responses to strong stimuli. These prolonged responses are mediated by long-lasting calcium-activated non-selective cationic current (Ican). In the current study we further examined the firing characteristics of these cells and their modulation by several neuromodulators. We found that AOB mitral cells display transient depolarizing afterpotentials (DAPs) following moderate firing. These DAPs are not found in MOB mitral cells that show instead robust hyperpolarizing afterpotentials. Unlike Ican, the DAPs of AOB mitral cells are activated by low levels of intracellular calcium and are relatively insensitive to flufenamic acid. Moreover, the cholinergic agonist carbachol exerts opposite effects on the persistent firing and DAPs of AOB mitral cells. We conclude that these phenomena are mediated by distinct biophysical mechanisms that may serve to mediate different types of information processing in the AOB at distinct brain states.

Calcium-activated sustained firing responses distinguish accessory from main olfactory bulb mitral cells.

Many mammals rely on pheromones for mediating social interactions. Recent studies indicate that both the main olfactory system (MOS) and accessory olfactory system (AOS) detect and process pheromonal stimuli, yet the functional difference between these two chemosensory systems remains unclear. We hypothesized that the main functional distinction between the MOS and AOS is the type of sensory information processing performed by each system. Here we compared the electrophysiological responses of mitral cells recorded from the accessory olfactory bulb (AOB) and main olfactory bulb (MOB) in acute mouse brain slices to various stimuli and found them markedly different. The response of MOB mitral cells to brief (0.1 ms, 1-100 V) stimulation of their sensory afferents remained transient regardless of stimulus strength, whereas sufficiently strong stimuli evoked sustained firing in AOB mitral cells lasting up to several minutes. Using EPSC-like current injections (10-100 pA, 10 ms rise time constant, 5 s decay time constant) in the presence of various synaptic blockers (picrotoxin, CGP55845, APV, DNQX, E4CPG, and MSPG), we demonstrated that this difference is attributable to distinct intrinsic properties of the two neuronal populations. The AOB sustained responses were found to be mediated by calcium-activated nonselective cationic current induced by transient intense firing. This current was found to be at least partially mediated by TRPM4 channels activated by calcium influx. We hypothesize that the sustained activity of the AOS induces a new sensory state in the animal, reflecting its social context.