Maternal gastrointestinal nematode infection alters hippocampal neuroimmunity, promotes synaptic plasticity, and improves resistance to direct infection in offspring.

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.

Akt and AMPK activators rescue hyperexcitability in neurons from patients with bipolar disorder.

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.

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons.

Background: Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Methods: Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. Results: We first identified specific non-coding variants in CTSB that drive the association with PD and are linked to changes in brain CTSB expression levels. Using iPSC-derived dopaminergic neurons we then find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. Moreover, in cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. Similarly, in midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition or knockout potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. Conclusions: The results of our genetic and functional studies indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Inhibition of LRRK2 kinase activity rescues deficits in striatal dopamine physiology in VPS35 p.D620N knock-in mice.

Dysregulation of dopamine neurotransmission profoundly affects motor, motivation and learning behaviors, and can be observed during the prodromal phase of Parkinson's disease (PD). However, the mechanism underlying these pathophysiological changes remains to be elucidated. Mutations in vacuolar protein sorting 35 (VPS35) and leucine-rich repeat kinase 2 (LRRK2) both lead to autosomal dominant PD, and VPS35 and LRRK2 may physically interact to govern the trafficking of synaptic cargos within the endo-lysosomal network in a kinase-dependent manner. To better understand the functional role of VPS35 and LRRK2 on dopamine physiology, we examined Vps35 haploinsufficient (Haplo) and Vps35 p.D620N knock-in (VKI) mice and how their behavior, dopamine kinetics and biochemistry are influenced by LRRK2 kinase inhibitors. We found Vps35 p.D620N significantly elevates LRRK2-mediated phosphorylation of Rab10, Rab12 and Rab29. In contrast, Vps35 haploinsufficiency reduces phosphorylation of Rab12. While striatal dopamine transporter (DAT) expression and function is similarly impaired in both VKI and Haplo mice, that physiology is normalized in VKI by treatment with the LRRK2 kinase inhibitor, MLi-2. As a corollary, VKI animals show a significant increase in amphetamine induced hyperlocomotion, compared to Haplo mice, that is also abolished by MLi-2. Taken together, these data show Vps35 p.D620N confers a gain-of-function with respect to LRRK2 kinase activity, and that VPS35 and LRRK2 functionally interact to regulate DAT function and striatal dopamine transmission.

The Parkinson's disease risk gene cathepsin B promotes fibrillar alpha-synuclein clearance, lysosomal function and glucocerebrosidase activity in dopaminergic neurons.

Variants in the CTSB gene encoding the lysosomal hydrolase cathepsin B (catB) are associated with increased risk of Parkinson's disease (PD). However, neither the specific CTSB variants driving these associations nor the functional pathways that link catB to PD pathogenesis have been characterized. CatB activity contributes to lysosomal protein degradation and regulates signaling processes involved in autophagy and lysosome biogenesis. Previous in vitro studies have found that catB can cleave monomeric and fibrillar alpha-synuclein, a key protein involved in the pathogenesis of PD that accumulates in the brains of PD patients. However, truncated synuclein isoforms generated by catB cleavage have an increased propensity to aggregate. Thus, catB activity could potentially contribute to lysosomal degradation and clearance of pathogenic alpha synuclein from the cell, but also has the potential of enhancing synuclein pathology by generating aggregation-prone truncations. Therefore, the mechanisms linking catB to PD pathophysiology remain to be clarified. Here, we conducted genetic analyses of the association between common and rare CTSB variants and risk of PD. We then used genetic and pharmacological approaches to manipulate catB expression and function in cell lines and induced pluripotent stem cell-derived dopaminergic neurons and assessed lysosomal activity and the handling of aggregated synuclein fibrils. We find that catB inhibition impairs autophagy, reduces glucocerebrosidase (encoded by GBA1) activity, and leads to an accumulation of lysosomal content. In cell lines, reduction of CTSB gene expression impairs the degradation of pre-formed alpha-synuclein fibrils, whereas CTSB gene activation enhances fibril clearance. In midbrain organoids and dopaminergic neurons treated with alpha-synuclein fibrils, catB inhibition potentiates the formation of inclusions which stain positively for phosphorylated alpha-synuclein. These results indicate that the reduction of catB function negatively impacts lysosomal pathways associated with PD pathogenesis, while conversely catB activation could promote the clearance of pathogenic alpha-synuclein.

Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture.

Long-term stable cell culture is a critical tool to better understand cell function. Most adherent cell culture models require a polymer substrate coating of poly-lysine or poly-ornithine for the cells to adhere and survive. However, polypeptide-based substrates are degraded by proteolysis and it remains a challenge to maintain healthy cell cultures for extended periods of time. Here, we report the development of an enhanced cell culture substrate based on a coating of dendritic polyglycerol amine (dPGA), a non-protein macromolecular biomimetic of poly-lysine, to promote the adhesion and survival of neurons in cell culture. We show that this new polymer coating provides enhanced survival, differentiation and long-term stability for cultures of primary neurons or neurons derived from human induced pluripotent stem cells (hiPSCs). Atomic force microscopy analysis provides evidence that greater nanoscale roughness contributes to the enhanced capacity of dPGA-coated surfaces to support cells in culture. We conclude that dPGA is a cytocompatible, functionally superior, easy to use, low cost and highly stable alternative to poly-cationic polymer cell culture substrate coatings such as poly-lysine and poly-ornithine. Summary statementHere, we describe a novel dendritic polyglycerol amine-based substrate coating, demonstrating superior performance compared to current polymer coatings for long-term culture of primary neurons and neurons derived from induced pluripotent stem cells.

Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition.

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.

Chronic lithium treatment alters the excitatory/ inhibitory balance of synaptic networks and reduces mGluR5-PKC signalling in mouse cortical neurons.

Background: Bipolar disorder is characterized by cyclical alternation between mania and depression, often comorbid with psychosis and suicide. Compared with other medications, the mood stabilizer lithium is the most effective treatment for the prevention of manic and depressive episodes. However, the pathophysiology of bipolar disorder and lithium's mode of action are yet to be fully understood. Evidence suggests a change in the balance of excitatory and inhibitory activity, favouring excitation in bipolar disorder. In the present study, we sought to establish a holistic understanding of the neuronal consequences of lithium exposure in mouse cortical neurons, and to identify underlying mechanisms of action. Methods: We used a range of technical approaches to determine the effects of acute and chronic lithium treatment on mature mouse cortical neurons. We combined RNA screening and biochemical and electrophysiological approaches with confocal immunofluorescence and live-cell calcium imaging. Results: We found that only chronic lithium treatment significantly reduced intracellular calcium flux, specifically by activating metabotropic glutamatergic receptor 5. This was associated with altered phosphorylation of protein kinase C and glycogen synthase kinase 3, reduced neuronal excitability and several alterations to synapse function. Consequently, lithium treatment shifts the excitatory­inhibitory balance toward inhibition. Limitations: The mechanisms we identified should be validated in future by similar experiments in whole animals and human neurons. Conclusion: Together, the results revealed how lithium dampens neuronal excitability and the activity of the glutamatergic network, both of which are predicted to be overactive in the manic phase of bipolar disorder. Our working model of lithium action enables the development of targeted strategies to restore the balance of overactive networks, mimicking the therapeutic benefits of lithium but with reduced toxicity.

Chronic and Acute Manipulation of Cortical Glutamate Transmission Induces Structural and Synaptic Changes in Co-cultured Striatal Neurons.

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.

A Critical LRRK at the Synapse? The Neurobiological Function and Pathophysiological Dysfunction of LRRK2.

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.

Neuron-autonomous susceptibility to induced synuclein aggregation is exacerbated by endogenous Lrrk2 mutations and ameliorated by Lrrk2 genetic knock-out.

Neuronal aggregates containing α-synuclein are a pathological hallmark of several degenerative diseases; including Parkinson's disease, Parkinson's disease with dementia and dementia with Lewy bodies. Understanding the process of α-synuclein aggregation, and discovering means of preventing it, may help guide therapeutic strategy and drug design. Recent advances provide tools to induce α-synuclein aggregation in neuronal cultures. Application of exogenous pre-formed fibrillar α-synuclein induces pathological phosphorylation and accumulation of endogenous α-synuclein, typical of that seen in disease. Genomic variability and mutations in α-synuclein and leucine-rich repeat kinase 2 proteins are the major genetic risk factors for Parkinson's disease. Reports demonstrate fibril-induced α-synuclein aggregation is increased in cells from leucine-rich repeat kinase 2 pathogenic mutant (G2019S) overexpressing mice, and variously decreased by leucine-rich repeat kinase 2 inhibitors. Elsewhere in vivo antisense knock-down of leucine-rich repeat kinase 2 protein has been shown to protect mice from fibril-induced α-synuclein aggregation, whereas kinase inhibition did not. To help bring clarity to this issue, we took a purely genetic approach in a standardized neuron-enriched culture, lacking glia. We compared fibril treatment of leucine-rich repeat kinase 2 germ-line knock-out, and G2019S germ-line knock-in, mouse cortical neuron cultures with those from littermates. We found leucine-rich repeat kinase 2 knock-out neurons are resistant to α-synuclein aggregation, which predominantly forms within axons, and may cause axonal fragmentation. Conversely, leucine-rich repeat kinase 2 knock-in neurons are more vulnerable to fibril-induced α-synuclein accumulation. Protection and resistance correlated with basal increases in a lysosome marker in knock-out, and an autophagy marker in knock-in cultures. The data add to a growing number of studies that argue leucine-rich repeat kinase 2 silencing, and potentially kinase inhibition, may be a useful therapeutic strategy against synucleinopathy.

Quantitative Profiling of Synuclein Species: Application to Transgenic Mouse Models of Parkinson's Disease.

INTRODUCTION: Improved analytical tools for detailed characterization of synucleins in pre-clinical models of Parkinson's disease (PD) and related synucleinopathies are needed. OBJECTIVE: Develop a multiple reaction monitoring (MRM) liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to quantify species-specific sequences and structural heterogeneity in soluble α- and ß-synucleins in brain tissue. METHODS: Using a proteolytic digestion workflow, the MRM LC-MS/MS method assayed six proteotypic peptides from the α-synuclein sequence; three unique to mouse or human α-synuclein and three conserved in α- and ß-synuclein. For quantification, we used labeled α-synuclein as the internal standard and an external calibration curve. As proof of concept, the synuclein LC-MS/MS method was applied to brain tissue specimens from M83 transgenic PD mice, which overexpresses human α-synuclein, relative to wild-type littermate controls. RESULTS: The synuclein MRM assay was linear over a wide concentration range (at least one order of magnitude). The assay had several advantages over ligand binding analytical methods, such as western blotting and enzyme-linked immunosorbent assays. These advantages included the ability to: quantify 1) total α-synuclein, 2) combined α- and ß-synucleins, 3) species-specific contributions to total α-synuclein (e.g., in mice expressing both mouse and human α-synuclein), and 4) identify peptide-specific profile differences that may reflect post-translational modifications, all within a single analysis. CONCLUSION: With improved and expanded analytical characteristics coupled with a streamlined sample preparation workflow, the quantitative synuclein profiling LC-MS/MS assay provides a versatile and efficient platform to characterize synuclein biology in pre-clinical models and the potential for application to human tissues and fluids.

The X-Linked Intellectual Disability Gene Zdhhc9 Is Essential for Dendrite Outgrowth and Inhibitory Synapse Formation.

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.

DNAJC13 p.Asn855Ser, implicated in familial parkinsonism, alters membrane dynamics of sorting nexin 1.

DNAJC13 (RME-8) is a core co-chaperone that facilitates membrane recycling and cargo sorting of endocytosed proteins. DNAJ/Hsp40 (heat shock protein 40) proteins are highly conserved throughout evolution and mediate the folding of nascent proteins, and the unfolding, refolding or degradation of misfolded proteins while assisting in associated-membrane translocation. DNAJC13 is one of five DNAJ 'C' class chaperone variants implicated in monogenic parkinsonism. Here we examine the effect of the DNAJC13 disease-linked mutation (p.Asn855Ser) on its interacting partners, focusing on sorting nexin 1 (SNX1) membrane dynamics in primary cortical neurons derived from a novel Dnajc13 p.Asn855Ser knock-in (DKI) mouse model. Dnajc13 p.Asn855Ser mutant and wild type protein expression were equivalent in mature heterozygous cultures (DIV21). While SNX1-positive puncta density, area, and WASH-retromer assembly were comparable between cultures derived from DKI and wild type littermates, the formation of SNX1-enriched tubules in DKI neuronal cultures was significantly increased. Thus, Dnajc13 p.Asn855Ser disrupts SNX1 membrane-tubulation and trafficking, analogous to results from RME-8 depletion studies. The data suggest the mutation confers a dominant-negative gain-of-function in RME-8. Implications for the pathogenesis of Parkinson's disease are discussed.

Altered dopamine release and monoamine transporters in Vps35 p.D620N knock-in mice.

Vacuolar protein sorting 35 (VPS35) is a core component of the retromer trimer required for endosomal membrane-associated protein trafficking. The discovery of a missense mutation, Vps35 p.D620N implicates retromer dysfunction in the pathogenesis of Parkinson's disease (PD). We have characterized a knock-in mouse with a Vps35 p.D620N substitution (hereafter referred to as VKI) at 3 months of age. Standardized behavioral testing did not observe overt movement disorder. Tyrosine hydroxylase (TH)-positive nigral neuron counts and terminal expression in striata were comparable across genotypes. Fast scan cyclic voltammetry revealed increased dopamine release in VKI striatal slices. While extracellular dopamine collected via striatal microdialysis of freely moving animals was comparable across genotypes, the ratio of dopamine metabolites to dopamine suggests increased dopamine turnover in VKI homozygous mice. Western blot of striatal proteins revealed a genotype-dependent decrease in dopamine transporter (DAT) along with an increase in vesicular monoamine transporter 2 (VMAT2), albeit independent of changes in other synaptic markers. The reduction in DAT was further supported by immunohistochemical analysis. The data show that the dopaminergic system of VKI mice is profoundly altered relative to wild-type littermates. We conclude early synaptic dysfunction contributes to age-related pathophysiology in the nigrostriatal system that may lead to parkinsonism in man.

HttQ111/+ Huntington's Disease Knock-in Mice Exhibit Brain Region-Specific Morphological Changes and Synaptic Dysfunction.

BACKGROUND: Successful disease-modifying therapy for Huntington's disease (HD) will require therapeutic intervention early in the pathogenic process. Achieving this goal requires identifying phenotypes that are proximal to the HTT CAG repeat expansion. OBJECTIVE: To use Htt CAG knock-in mice, precise genetic replicas of the HTT mutation in patients, as models to study proximal disease events. METHODS: Using cohorts of B6J.HttQ111/+ mice from 2 to 18 months of age, we analyzed pathological markers, including immunohistochemistry, brain regional volumes and cortical thickness, CAG instability, electron microscopy of striatal synapses, and acute slice electrophysiology to record glutamatergic transmission at striatal synapses. We also incorporated a diet perturbation paradigm for some of these analyses. RESULTS: B6J.HttQ111/+ mice did not exhibit significant neurodegeneration or gliosis but revealed decreased striatal DARPP-32 as well as subtle but regional-specific changes in brain volumes and cortical thickness that parallel those in HD patients. Ultrastructural analyses of the striatum showed reduced synapse density, increased postsynaptic density thickness and increased synaptic cleft width. Acute slice electrophysiology showed alterations in spontaneous AMPA receptor-mediated postsynaptic currents, evoked NMDA receptor-mediated excitatory postsynaptic currents, and elevated extrasynaptic NMDA currents. Diet influenced cortical thickness, but did not impact somatic CAG expansion, nor did it show any significant interaction with genotype on immunohistochemical, brain volume or cortical thickness measures. CONCLUSIONS: These data show that a single HttQ111 allele is sufficient to elicit brain region-specific morphological changes and early neuronal dysfunction, highlighting an insidious disease process already apparent in the first few months of life.

Initial elevations in glutamate and dopamine neurotransmission decline with age, as does exploratory behavior, in LRRK2 G2019S knock-in mice.

LRRK2 mutations produce end-stage Parkinson's disease (PD) with reduced nigrostriatal dopamine, whereas, asymptomatic carriers have increased dopamine turnover and altered brain connectivity. LRRK2 pathophysiology remains unclear, but reduced dopamine and mitochondrial abnormalities occur in aged G2019S mutant knock-in (GKI) mice. Conversely, cultured GKI neurons exhibit increased synaptic transmission. We assessed behavior and synaptic glutamate and dopamine function across a range of ages. Young GKI mice exhibit more vertical exploration, elevated glutamate and dopamine transmission, and aberrant D2-receptor responses. These phenomena decline with age, but are stable in littermates. In young GKI mice, dopamine transients are slower, independent of dopamine transporter (DAT), increasing the lifetime of extracellular dopamine. Slowing of dopamine transients is observed with age in littermates, suggesting premature ageing of dopamine synapses in GKI mice. Thus, GKI mice exhibit early, but declining, synaptic and behavioral phenotypes, making them amenable to investigation of early pathophysiological, and later parkinsonian-like, alterations. This model will prove valuable in efforts to develop neuroprotection for PD.

DNM3 and genetic modifiers of age of onset in LRRK2 Gly2019Ser parkinsonism: a genome-wide linkage and association study.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) mutation 6055G→A (Gly2019Ser) accounts for roughly 1% of patients with Parkinson's disease in white populations, 13-30% in Ashkenazi Jewish populations, and 30-40% in North African Arab-Berber populations, although age of onset is variable. Some carriers have early-onset parkinsonism, whereas others remain asymptomatic despite advanced age. We aimed to use a genome-wide approach to identify genetic variability that directly affects LRRK2 Gly2019Ser penetrance. METHODS: Between 2006 and 2012, we recruited Arab-Berber patients with Parkinson's disease and their family members (aged 18 years or older) at the Mongi Ben Hamida National Institute of Neurology (Tunis, Tunisia). Patients with Parkinson's disease were diagnosed by movement disorder specialists in accordance with the UK Parkinson's Disease Society Brain Bank criteria, without exclusion of familial parkinsonism. LRRK2 carrier status was confirmed by Sanger sequencing or TaqMan SNP assays-on-demand. We did genome-wide linkage analysis using data from multi-incident Arab-Berber families with Parkinson's disease and LRRK2 Gly2019Ser (with both affected and unaffected family members). We assessed Parkinson's disease age of onset both as a categorical variable (dichotomised by median onset) and as a quantitative trait. We used data from another cohort of unrelated Tunisian LRRK2 Gly2019Ser carriers for subsequent locus-specific genotyping and association analyses. Whole-genome sequencing in a subset of 14 unrelated Arab-Berber individuals who were LRRK2 Gly2019Ser carriers (seven with early-onset disease and seven elderly unaffected individuals) subsequently informed imputation and haplotype analyses. We replicated the findings in separate series of LRRK2 Gly2019Ser carriers originating from Algeria, France, Norway, and North America. We also investigated associations between genotype, gene, and protein expression in human striatal tissues and murine LRRK2 Gly2019Ser cortical neurons. FINDINGS: Using data from 41 multi-incident Arab-Berber families with Parkinson's disease and LRRK2 Gly2019Ser (150 patients and 103 unaffected family members), we identified significant linkage on chromosome 1q23.3 to 1q24.3 (non-parametric logarithm of odds score 2·9, model-based logarithm of odds score 4·99, θ=0 at D1S2768). In a cohort of unrelated Arab-Berber LRRK2 Gly2019Ser carriers, subsequent association mapping within the linkage region suggested genetic variability within DNM3 as an age-of-onset modifier of disease (n=232; rs2421947; haplotype p=1·07 × 10-7). We found that DNM3 rs2421947 was a haplotype tag for which the median onset of LRRK2 parkinsonism in GG carriers was 12·5 years younger than that of CC carriers (Arab-Berber cohort, hazard ratio [HR] 1·89, 95% CI 1·20-2·98). Replication analyses in separate series from Algeria, France, Norway, and North America (n=263) supported this finding (meta-analysis HR 1·61, 95% CI 1·15-2·27, p=0·02). In human striatum, DNM3 expression varied as a function of rs2421947 genotype, and dynamin-3 localisation was perturbed in murine LRRK2 Gly2019Ser cortical neurons. INTERPRETATION: Genetic variability in DNM3 modifies age of onset for LRRK2 Gly2019Ser parkinsonism and informs disease-relevant translational neuroscience. Our results could be useful in genetic counselling for carriers of this mutation and in clinical trial design. FUNDING: The Canada Excellence Research Chairs (CERC), Leading Edge Endowment Fund (LEEF), Don Rix BC Leadership Chair in Genetic Medicine, National Institute on Aging, National Institute of Neurological Disorders and Stroke, the Michael J Fox Foundation, Mayo Foundation, the Roger de Spoelberch Foundation, and GlaxoSmithKline.

G2019S-LRRK2 Expression Augments α-Synuclein Sequestration into Inclusions in Neurons.

UNLABELLED: Pathologic inclusions define α-synucleinopathies that include Parkinson's disease (PD). The most common genetic cause of PD is the G2019S LRRK2 mutation that upregulates LRRK2 kinase activity. However, the interaction between α-synuclein, LRRK2, and the formation of α-synuclein inclusions remains unclear. Here, we show that G2019S-LRRK2 expression, in both cultured neurons and dopaminergic neurons in the rat substantia nigra pars compact, increases the recruitment of endogenous α-synuclein into inclusions in response to α-synuclein fibril exposure. This results from the expression of mutant G2019S-LRRK2, as overexpression of WT-LRRK2 not only does not increase formation of inclusions but reduces their abundance. In addition, treatment of primary mouse neurons with LRRK2 kinase inhibitors, PF-06447475 and MLi-2, blocks G2019S-LRRK2 effects, suggesting that the G2019S-LRRK2 potentiation of inclusion formation depends on its kinase activity. Overexpression of G2019S-LRRK2 slightly increases, whereas WT-LRRK2 decreases, total levels of α-synuclein. Knockdown of total α-synuclein with potent antisense oligonucleotides substantially reduces inclusion formation in G2019S-LRRK2-expressing neurons, suggesting that LRRK2 influences α-synuclein inclusion formation by altering α-synuclein levels. These findings support the hypothesis that G2019S-LRRK2 may increase the progression of pathological α-synuclein inclusions after the initial formation of α-synuclein pathology by increasing a pool of α-synuclein that is more susceptible to forming inclusions. SIGNIFICANCE STATEMENT: α-Synuclein inclusions are found in the brains of patients with many different neurodegenerative diseases. Point mutation, duplication, or triplication of the α-synuclein gene can all cause Parkinson's disease (PD). The G2019S mutation in LRRK2 is the most common known genetic cause of PD. The interaction between G2019S-LRRK2 and α-synuclein may uncover new mechanisms and targets for neuroprotection. Here, we show that expression of G2019S-LRRK2 increases α-synuclein mobility and enhances aggregation of α-synuclein in primary cultured neurons and in dopaminergic neurons of the substantia nigra pars compacta, a susceptible brain region in PD. Potent LRRK2 kinase inhibitors, which are being developed for clinical use, block the increased α-synuclein aggregation in G2019S-LRRK2-expressing neurons. These results demonstrate that α-synuclein inclusion formation in neurons can be blocked and that novel therapeutic compounds targeting this process by inhibiting LRRK2 kinase activity may slow progression of PD-associated pathology.

Changes in Dopamine Signalling Do Not Underlie Aberrant Hippocampal Plasticity in a Mouse Model of Huntington's Disease.

Altered dopamine receptor labelling has been demonstrated in presymptomatic and symptomatic Huntington's disease (HD) gene carriers, indicating that alterations in dopaminergic signalling are an early event in HD. We have previously described early alterations in synaptic transmission and plasticity in both the cortex and hippocampus of the R6/1 mouse model of Huntington's disease. Deficits in cortical synaptic plasticity were associated with altered dopaminergic signalling and could be reversed by D1- or D2-like dopamine receptor activation. In light of these findings we here investigated whether defects in dopamine signalling could also contribute to the marked alteration in hippocampal synaptic function. To this end we performed dopamine receptor labelling and pharmacology in the R6/1 hippocampus and report a marked, age-dependent elevation of hippocampal D1 and D2 receptor labelling in R6/1 hippocampal subfields. Yet, pharmacological inhibition or activation of D1- or D2-like receptors did not modify the aberrant synaptic plasticity observed in R6/1 mice. These findings demonstrate that global perturbations to dopamine receptor expression do occur in HD transgenic mice, similarly in HD gene carriers and patients. However, the direction of change and the lack of effect of dopaminergic pharmacological agents on synaptic function demonstrate that the perturbations are heterogeneous and region-specific, a finding that may explain the mixed results of dopamine therapy in HD.