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Abstract The developing brain is vulnerable to maternal bacterial and viral infections which induce strong inflammatory responses in the mother that are mimicked in the offspring brain, resulting in irreversible neurodevelopmental defects, and associated cognitive and behavioural impairments. In contrast, infection during pregnancy and lactation with the immunoregulatory murine intestinal nematode, Heligmosomoides bakeri, upregulates expression of genes associated with long-term potentiation (LTP) of synaptic networks in the brain of neonatal uninfected offspring, and enhances spatial memory in uninfected juvenile offspring. As the hippocampus is involved in spatial navigation and sensitive to immune events during development, here we assessed hippocampal gene expression, LTP, and neuroimmunity in 3-week-old uninfected offspring born to H. bakeri infected mothers. Further, as maternal immunity shapes the developing immune system, we assessed the impact of maternal H. bakeri infection on the ability of offspring to resist direct infection. In response to maternal infection, we found an enhanced propensity to induce LTP at Schaffer collateral synapses, consistent with RNA-seq data indicating accelerated development of glutamatergic synapses in uninfected offspring, relative to those from uninfected mothers. Hippocampal RNA-seq analysis of offspring of infected mothers revealed increased expression of genes associated with neurogenesis, gliogenesis, and myelination. Furthermore, maternal infection improved resistance to direct infection of H. bakeri in offspring, correlated with transfer of parasite-specific IgG1 to their serum. Hippocampal immunohistochemistry and gene expression suggest Th2/Treg biased neuroimmunity in offspring, recapitulating peripheral immunoregulation of H. bakeri infected mothers. These findings indicate maternal H. bakeri infection during pregnancy and lactation alters peripheral and neural immunity in uninfected offspring, in a manner that accelerates neural maturation to promote hippocampal LTP, and upregulates the expression of genes associated with neurogenesis, gliogenesis, and myelination.

Summary: Background: Bipolar disorder (BD) is a multifactorial psychiatric illness affecting ∼1% of the global adult population. Lithium (Li), is the most effective mood stabilizer for BD but works only for a subset of patients and its mechanism of action remains largely elusive. Methods: In the present study, we used iPSC-derived neurons from patients with BD who are responsive (LR) or not (LNR) to lithium. Combined electrophysiology, calcium imaging, biochemistry, transcriptomics, and phosphoproteomics were employed to provide mechanistic insights into neuronal hyperactivity in BD, investigate Li's mode of action, and identify alternative treatment strategies. Findings: We show a selective rescue of the neuronal hyperactivity phenotype by Li in LR neurons, correlated with changes to Na+ conductance. Whole transcriptome sequencing in BD neurons revealed altered gene expression pathways related to glutamate transmission, alterations in cell signalling and ion transport/channel activity. We found altered Akt signalling as a potential therapeutic effect of Li in LR neurons from patients with BD, and that Akt activation mimics Li effect in LR neurons. Furthermore, the increased neural network activity observed in both LR & LNR neurons from patients with BD were reversed by AMP-activated protein kinase (AMPK) activation. Interpretation: These results suggest potential for new treatment strategies in BD, such as Akt activators in LR cases, and the use of AMPK activators for LNR patients with BD. Funding: Supported by funding from ERA PerMed, Bell Brain Canada Mental Research Program and Brain & Behavior Research Foundation.

Abstract Vacuolar protein sorting 35 (VPS35) regulates neurotransmitter receptor recycling from endosomes. A missense mutation (D620N) in VPS35 leads to autosomal-dominant, late-onset Parkinson’s disease. Here, we study the basic neurobiology of VPS35 and Parkinson’s disease mutation effects in the D620N knock-in mouse and the effect of leucine-rich repeat kinase 2 (LRRK2) inhibition on synaptic phenotypes. The study was conducted using a VPS35 D620N knock-in mouse that expresses VPS35 at endogenous levels. Protein levels, phosphorylation states, and binding ratios in brain lysates from knock-in mice and wild-type littermates were assayed by co-immunoprecipitation and western blot. Dendritic protein co-localization, AMPA receptor surface expression, synapse density, and glutamatergic synapse activity in primary cortical cultures from knock-in and wild-type littermates were assayed using immunocytochemistry and whole-cell patch clamp electrophysiology. In brain tissue, we confirm VPS35 forms complexes with LRRK2 and AMPA-type glutamate receptor GluA1 subunits, in addition to NMDA-type glutamate receptor GluN1 subunits and D2-type dopamine receptors. Receptor and LRRK2 binding was unaltered in D620N knock-in mice, but we confirm the mutation results in reduced binding of VPS35 with WASH complex member FAM21, and increases phosphorylation of the LRRK2 kinase substrate Rab10, which is reversed by LRRK2 kinase inhibition in vivo. In cultured cortical neurons from knock-in mice, pRab10 is also increased, and reversed by LRRK2 inhibition. The mutation also results in increased endosomal recycling protein cluster density (VPS35-FAM21 co-clusters and Rab11 clusters), glutamate transmission, and GluA1 surface expression. LRRK2 kinase inhibition, which reversed Rab10 hyper-phosphorylation, did not rescue elevated glutamate release or surface GluA1 expression in knock-in neurons, but did alter AMPAR traffic in wild-type cells. The results improve our understanding of the cell biology of VPS35, and the consequences of the D620N mutation in developing neuronal networks. Together the data support a chronic synaptopathy model for latent neurodegeneration, providing phenotypes and candidate pathophysiological stresses that may drive eventual transition to late-stage parkinsonism in VPS35 PD. The study demonstrates the VPS35 mutation has effects that are independent of ongoing LRRK2 kinase activity, and that LRRK2 kinase inhibition alters basal physiology of glutamate synapses in vitro.

In contrast to the prenatal topographic development of sensory cortices, striatal circuit organization is slow and requires the functional maturation of cortical and thalamic excitatory inputs throughout the first postnatal month. While mechanisms regulating synapse development and plasticity are quite well described at excitatory synapses of glutamatergic neurons in the neocortex, comparatively little is known of how this translates to glutamate synapses onto GABAergic neurons in the striatum. Here we investigate excitatory striatal synapse plasticity in an in vitro system, where glutamate can be studied in isolation from dopamine and other neuromodulators. We examined pre-and post-synaptic structural and functional plasticity in GABAergic striatal spiny projection neurons (SPNs), co-cultured with glutamatergic cortical neurons. After synapse formation, medium-term (24 h) TTX silencing increased the density of filopodia, and modestly decreased dendritic spine density, when assayed at 21 days in vitro (DIV). Spine reductions appeared to require residual spontaneous activation of ionotropic glutamate receptors. Conversely, chronic (14 days) TTX silencing markedly reduced spine density without any observed increase in filopodia density. Time-dependent, biphasic changes to the presynaptic marker Synapsin-1 were also observed, independent of residual spontaneous activity. Acute silencing (3 h) did not affect presynaptic markers or postsynaptic structures. To induce rapid, activity-dependent plasticity in striatal neurons, a chemical NMDA receptor-dependent “long-term potentiation (LTP)” paradigm was employed. Within 30 min, this increased spine and GluA1 cluster densities, and the percentage of spines containing GluA1 clusters, without altering the presynaptic signal. The results demonstrate that the growth and pruning of dendritic protrusions is an active process, requiring glutamate receptor activity in striatal projection neurons. Furthermore, NMDA receptor activation is sufficient to drive glutamatergic structural plasticity in SPNs, in the absence of dopamine or other neuromodulators.

Since the discovery of LRRK2 mutations causal to Parkinson’s disease (PD) in the early 2000s, the LRRK2 protein has been implicated in a plethora of cellular processes in which pathogenesis could occur, yet its physiological function remains elusive. The development of genetic models of LRRK2 PD has helped identify the etiological and pathophysiological underpinnings of the disease, and may identify early points of intervention. An important role for LRRK2 in synaptic function has emerged in recent years, which links LRRK2 to other genetic forms of PD, most notably those caused by mutations in the synaptic protein α-synuclein. This point of convergence may provide useful clues as to what drives dysfunction in the basal ganglia circuitry and eventual death of substantia nigra (SN) neurons. Here, we discuss the evolution and current state of the literature placing LRRK2 at the synapse, through the lens of knock-out, overexpression, and knock-in animal models. We hope that a deeper understanding of LRRK2 neurobiology, at the synapse and beyond, will aid the eventual development of neuroprotective interventions for PD, and the advancement of useful treatments in the interim.

Summary: Palmitoylation is a reversible post-translational lipid modification that facilitates vesicular transport and subcellular localization of modified proteins. This process is catalyzed by ZDHHC enzymes that are implicated in several neurological and neurodevelopmental disorders. Loss-of-function mutations in ZDHHC9 have been identified in patients with X-linked intellectual disability (XLID) and associated with increased epilepsy risk. Loss of Zdhhc9 function in hippocampal cultures leads to shorter dendritic arbors and fewer inhibitory synapses, altering the ratio of excitatory-to-inhibitory inputs formed onto Zdhhc9-deficient cells. While Zdhhc9 promotes dendrite outgrowth through the palmitoylation of the GTPase Ras, it promotes inhibitory synapse formation through the palmitoylation of another GTPase, TC10. Zdhhc9 knockout mice exhibit seizure-like activity together with increased frequency and amplitude of both spontaneous and miniature excitatory and inhibitory postsynaptic currents. These findings present a plausible mechanism for how the loss of ZDHHC9 function may contribute to XLID and epilepsy. : Shimell et al. demonstrate that the palmitoylating enzyme Zdhhc9 controls dendritic growth and maintains excitatory/inhibitory synapse balance through distinct substrates. Loss of Zdhhc9 increases network excitability and seizure activity in accordance with Zdhhc9’s association with X-linked intellectual disability and epilepsy. Keywords: Zdhhc9, palmitoylation, neuron morphology, synapse, hippocampal culture, X-linked intellectual disability, dendrite growth, dendrite retraction, Ras GTPase, TC10 GTPase, epilepsy

We recently reported evidence for disturbed synaptic versus extrasynaptic NMDAR transmission in the early pathogenesis of Huntington's disease (HD), a late-onset neurodegenerative disorder caused by CAG repeat expansion in the gene encoding huntingtin. Studies in glutamatergic cells indicate that synaptic NMDAR transmission increases phosphorylated cyclic-AMP response element binding protein (pCREB) levels and drives neuroprotective gene transcription, whereas extrasynaptic NMDAR activation reduces pCREB and promotes cell death. By generating striatal and cortical neuronal co-cultures to investigate the glutamatergic innervation of striatal neurons, we demonstrate that dichotomous synaptic and extrasynaptic NMDAR signaling also occurs in GABAergic striatal medium-sized spiny neurons (MSNs), which are acutely vulnerable in HD. Further, we show that wild-type (WT) and HD transgenic YAC128 MSNs co-cultured with cortical cells have similar levels of glutamatergic synapses, synaptic NMDAR currents and synaptic GluN2B and GluN2A subunit-containing NMDARs. However, NMDAR whole-cell, and especially extrasynaptic, current is elevated in YAC128 MSNs. Moreover, GluN2B subunit-containing NMDAR surface expression is markedly increased, irrespective of whether or not the co-cultured cortical cells express mutant huntingtin. The data suggest that MSN cell-autonomous increases in extrasynaptic NMDARs are driven by the HD mutation. Consistent with these results, we find that extrasynaptic NMDAR-induced pCREB reductions and apoptosis are also augmented in YAC128 MSNs. Moreover, both NMDAR-mediated apoptosis and CREB-off signaling are blocked by co-application of either memantine or the GluN2B subunit-selective antagonist ifenprodil in YAC128 MSNs. GluN2A-subunit-selective concentrations of the antagonist NVP-AAM077 did not reduce cell death in either genotype. Cortico-striatal co-cultures provide an in vitro model system in which to better investigate striatal neuronal dysfunction in disease than mono-cultured striatal cells. Results from the use of this system, which partially recapitulates the cortico-striatal circuit and is amenable to acute genetic and pharmacological manipulations, suggest that pathophysiological NMDAR signaling is an intrinsic frailty in HD MSNs that can be successfully targeted by pharmacological interventions.

Huntington disease (HD) is a dominantly inherited neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the protein huntingtin (htt). Previous studies have shown enhanced N-methyl-d-aspartate (NMDA)-induced excitotoxicity in neuronal models of HD, mediated in part by increased NMDA receptor (NMDAR) GluN2B subunit binding with the postsynaptic density protein-95 (PSD-95). In cultured hippocampal neurons, the NMDAR-activated p38 Mitogen-activated Protein Kinase (MAPK) death pathway is disrupted by a peptide (Tat-NR2B9c) that uncouples GluN2B from PSD-95, whereas NMDAR-mediated activation of c-Jun N-terminal Kinase (JNK) MAPK is PSD-95-independent. To investigate the mechanism by which Tat-NR2B9c protects striatal medium spiny neurons (MSNs) from mutant htt (mhtt)-enhanced NMDAR toxicity, we compared striatal tissue and cultured MSNs from presymptomatic yeast artificial chromosome (YAC) mice expressing htt with 128 polyQ (YAC128) to those from YAC18 and/or WT mice as controls. Similar to the previously published shift of GluN2B-containing NMDARs to extrasynaptic sites, we found increased PSD-95 localization as well as elevated PSD-95-GluN2B interactions in the striatal non-PSD (extrasynaptic) fraction from YAC128 mice. Notably, basal levels of both activated p38 and JNK MAPKs were elevated in the YAC128 striatum. NMDA stimulation of acute slices increased activation of p38 and JNK in WT and YAC128 striatum, but Tat-NR2B9c pretreatment reduced only the p38 activation in YAC128. In cultured MSNs, p38 MAPK inhibition reduced YAC128 NMDAR-mediated cell death to WT levels, and occluded the Tat-NR2B9c peptide protective effect; in contrast, inhibition of JNK had a similar protective effect in cultured MSNs from both WT and YAC128 mice. Our results suggest that altered activation of p38 MAPK contributes to mhtt enhancement of GluN2B/PSD-95 toxic signaling.