Cell type-specific functions of Alzheimer's disease endocytic risk genes.

Endocytosis is a key cellular pathway required for the internalization of cellular nutrients, lipids and receptor-bound cargoes. It is also critical for the recycling of cellular components, cellular trafficking and membrane dynamics. The endocytic pathway has been consistently implicated in Alzheimer's disease (AD) through repeated genome-wide association studies and the existence of rare coding mutations in endocytic genes. BIN1 and PICALM are two of the most significant late-onset AD risk genes after APOE and are both key to clathrin-mediated endocytic biology. Pathological studies also demonstrate that endocytic dysfunction is an early characteristic of late-onset AD, being seen in the prodromal phase of the disease. Different cell types of the brain have specific requirements of the endocytic pathway. Neurons require efficient recycling of synaptic vesicles and microglia use the specialized form of endocytosis-phagocytosis-for their normal function. Therefore, disease-associated changes in endocytic genes will have varied impacts across different cell types, which remains to be fully explored. Given the genetic and pathological evidence for endocytic dysfunction in AD, understanding how such changes and the related cell type-specific vulnerabilities impact normal cellular function and contribute to disease is vital and could present novel therapeutic opportunities. This article is part of a discussion meeting issue 'Understanding the endo-lysosomal network in neurodegeneration'.

Single-cell spatial transcriptomic and translatomic profiling of dopaminergic neurons in health, aging, and disease.

The brain is spatially organized and contains unique cell types, each performing diverse functions and exhibiting differential susceptibility to neurodegeneration. This is exemplified in Parkinson's disease with the preferential loss of dopaminergic neurons of the substantia nigra pars compacta. Using a Parkinson's transgenic model, we conducted a single-cell spatial transcriptomic and dopaminergic neuron translatomic analysis of young and old mouse brains. Through the high resolving capacity of single-cell spatial transcriptomics, we provide a deep characterization of the expression features of dopaminergic neurons and 27 other cell types within their spatial context, identifying markers of healthy and aging cells, spanning Parkinson's relevant pathways. We integrate gene enrichment and genome-wide association study data to prioritize putative causative genes for disease investigation, identifying CASR as a regulator of dopaminergic calcium handling. These datasets represent the largest public resource for the investigation of spatial gene expression in brain cells in health, aging, and disease.

Tau depletion in human neurons mitigates Aß-driven toxicity.

Alzheimer's disease (AD) is an age-related neurodegenerative condition and the most common type of dementia, characterised by pathological accumulation of extracellular plaques and intracellular neurofibrillary tangles that mainly consist of amyloid-ß (Aß) and hyperphosphorylated tau aggregates, respectively. Previous studies in mouse models with a targeted knock-out of the microtubule-associated protein tau (Mapt) gene demonstrated that Aß-driven toxicity is tau-dependent. However, human cellular models with chronic tau lowering remain unexplored. In this study, we generated stable tau-depleted human induced pluripotent stem cell (iPSC) isogenic panels from two healthy individuals using CRISPR-Cas9 technology. We then differentiated these iPSCs into cortical neurons in vitro in co-culture with primary rat cortical astrocytes before conducting electrophysiological and imaging experiments for a wide range of disease-relevant phenotypes. Both AD brain derived and recombinant Aß were used in this study to elicit toxic responses from the iPSC-derived cortical neurons. We showed that tau depletion in human iPSC-derived cortical neurons caused considerable reductions in neuronal activity without affecting synaptic density. We also observed neurite outgrowth impairments in two of the tau-depleted lines used. Finally, tau depletion protected neurons from adverse effects by mitigating the impact of exogenous Aß-induced hyperactivity, deficits in retrograde axonal transport of mitochondria, and neurodegeneration. Our study established stable human iPSC isogenic panels with chronic tau depletion from two healthy individuals. Cortical neurons derived from these iPSC lines showed that tau is essential in Aß-driven hyperactivity, axonal transport deficits, and neurodegeneration, consistent with studies conducted in Mapt-/- mouse models. These findings highlight the protective effects of chronic tau lowering strategies in AD pathogenesis and reinforce the potential in clinical settings. The tau-depleted human iPSC models can now be applied at scale to investigate the involvement of tau in disease-relevant pathways and cell types.

Inhibition of striatal dopamine release by the L-type calcium channel inhibitor isradipine co-varies with risk factors for Parkinson's.

Ca2+ entry into nigrostriatal dopamine (DA) neurons and axons via L-type voltage-gated Ca2+ channels (LTCCs) contributes, respectively, to pacemaker activity and DA release and has long been thought to contribute to vulnerability to degeneration in Parkinson's disease. LTCC function is greater in DA axons and neurons from substantia nigra pars compacta than from ventral tegmental area, but this is not explained by channel expression level. We tested the hypothesis that LTCC control of DA release is governed rather by local mechanisms, focussing on candidate biological factors known to operate differently between types of DA neurons and/or be associated with their differing vulnerability to parkinsonism, including biological sex, α-synuclein, DA transporters (DATs) and calbindin-D28k (Calb1). We detected evoked DA release ex vivo in mouse striatal slices using fast-scan cyclic voltammetry and assessed LTCC support of DA release by detecting the inhibition of DA release by the LTCC inhibitors isradipine or CP8. Using genetic knockouts or pharmacological manipulations, we identified that striatal LTCC support of DA release depended on multiple intersecting factors, in a regionally and sexually divergent manner. LTCC function was promoted by factors associated with Parkinsonian risk, including male sex, α-synuclein, DAT and a dorsolateral co-ordinate, but limited by factors associated with protection, that is, female sex, glucocerebrosidase activity, Calb1 and ventromedial co-ordinate. Together, these data show that LTCC function in DA axons and isradipine effect are locally governed and suggest they vary in a manner that in turn might impact on, or reflect, the cellular stress that leads to parkinsonian degeneration.

Mitochondrial dysfunction and mitophagy defects in LRRK2-R1441C Parkinson's disease models.

Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation.

Assaying Microglia Functions In Vitro.

Microglia, the main immune modulators of the central nervous system, have key roles in both the developing and adult brain. These functions include shaping healthy neuronal networks, carrying out immune surveillance, mediating inflammatory responses, and disposing of unwanted material. A wide variety of pathological conditions present with microglia dysregulation, highlighting the importance of these cells in both normal brain function and disease. Studies into microglial function in the context of both health and disease thus have the potential to provide tremendous insight across a broad range of research areas. In vitro culture of microglia, using primary cells, cell lines, or induced pluripotent stem cell derived microglia, allows researchers to generate reproducible, robust, and quantifiable data regarding microglia function. A broad range of assays have been successfully developed and optimised for characterizing microglial morphology, mediation of inflammation, endocytosis, phagocytosis, chemotaxis and random motility, and mediation of immunometabolism. This review describes the main functions of microglia, compares existing protocols for measuring these functions in vitro, and highlights common pitfalls and future areas for development. We aim to provide a comprehensive methodological guide for researchers planning to characterise microglial functions within a range of contexts and in vitro models.

ß-synuclein potentiates synaptic vesicle dopamine uptake and rescues dopaminergic neurons from MPTP-induced death in the absence of other synucleins.

Synucleins, a family of three proteins highly expressed in neurons, are predominantly known for the direct involvement of α-synuclein in the etiology and pathogenesis of Parkinson's and certain other neurodegenerative diseases, but their precise physiological functions are still not fully understood. Previous studies have demonstrated the importance of α-synuclein as a modulator of various mechanisms implicated in chemical neurotransmission, but information concerning the involvement of other synuclein family members, ß-synuclein and γ-synuclein, in molecular processes within presynaptic terminals is limited. Here, we demonstrated that the vesicular monoamine transporter 2-dependent dopamine uptake by synaptic vesicles isolated from the striatum of mice lacking ß-synuclein is significantly reduced. Reciprocally, reintroduction, either in vivo or in vitro, of ß-synuclein but not α-synuclein or γ-synuclein improves uptake by triple α/ß/γ-synuclein-deficient striatal vesicles. We also showed that the resistance of dopaminergic neurons of the substantia nigra pars compacta to subchronic administration of the Parkinson's disease-inducing prodrug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine depends on the presence of ß-synuclein but only when one or both other synucleins are absent. Furthermore, proteomic analysis of synuclein-deficient synaptic vesicles versus those containing only ß-synuclein revealed differences in their protein compositions. We suggest that the observed potentiation of dopamine uptake by ß-synuclein might be caused by different protein architecture of the synaptic vesicles. It is also feasible that such structural changes improve synaptic vesicle sequestration of 1-methyl-4-phenylpyridinium, a toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which would explain why dopaminergic neurons expressing ß-synuclein and lacking α-synuclein and/or γ-synuclein are resistant to this neurotoxin.

Striatal Dopamine Transporter Function Is Facilitated by Converging Biology of α-Synuclein and Cholesterol.

Striatal dopamine transporters (DAT) powerfully regulate dopamine signaling, and can contribute risk to degeneration in Parkinson's disease (PD). DATs can interact with the neuronal protein α-synuclein, which is associated with the etiology and molecular pathology of idiopathic and familial PD. Here, we tested whether DAT function in governing dopamine (DA) uptake and release is modified in a human-α-synuclein-overexpressing (SNCA-OVX) transgenic mouse model of early PD. Using fast-scan cyclic voltammetry (FCV) in ex vivo acute striatal slices to detect DA release, and biochemical assays, we show that several aspects of DAT function are promoted in SNCA-OVX mice. Compared to background control α-synuclein-null mice (Snca-null), the SNCA-OVX mice have elevated DA uptake rates, and more pronounced effects of DAT inhibitors on evoked extracellular DA concentrations ([DA]o) and on short-term plasticity (STP) in DA release, indicating DATs play a greater role in limiting DA release and in driving STP. We found that DAT membrane levels and radioligand binding sites correlated with α-synuclein level. Furthermore, DAT function in Snca-null and SNCA-OVX mice could also be promoted by applying cholesterol, and using Tof-SIMS we found genotype-differences in striatal lipids, with lower striatal cholesterol in SNCA-OVX mice. An inhibitor of cholesterol efflux transporter ABCA1 or a cholesterol chelator in SNCA-OVX mice reduced the effects of DAT-inhibitors on evoked [DA]o. Together these data indicate that human α-synuclein in a mouse model of PD promotes striatal DAT function, in a manner supported by extracellular cholesterol, suggesting converging biology of α-synuclein and cholesterol that regulates DAT function and could impact DA function and PD pathophysiology.

GABA uptake transporters support dopamine release in dorsal striatum with maladaptive downregulation in a parkinsonism model.

Striatal dopamine (DA) is critical for action and learning. Recent data show that DA release is under tonic inhibition by striatal GABA. Ambient striatal GABA tone on striatal projection neurons can be determined by plasma membrane GABA uptake transporters (GATs) located on astrocytes and neurons. However, whether striatal GATs and astrocytes determine DA output are unknown. We reveal that DA release in mouse dorsolateral striatum, but not nucleus accumbens core, is governed by GAT-1 and GAT-3. These GATs are partly localized to astrocytes, and are enriched in dorsolateral striatum compared to accumbens core. In a mouse model of early parkinsonism, GATs are downregulated, tonic GABAergic inhibition of DA release augmented, and nigrostriatal GABA co-release attenuated. These data define previously unappreciated and important roles for GATs and astrocytes in supporting DA release in striatum, and reveal a maladaptive plasticity in early parkinsonism that impairs DA output in vulnerable striatal regions.

CLR01 protects dopaminergic neurons in vitro and in mouse models of Parkinson's disease.

Parkinson's disease (PD) affects millions of patients worldwide and is characterized by alpha-synuclein aggregation in dopamine neurons. Molecular tweezers have shown high potential as anti-aggregation agents targeting positively charged residues of proteins undergoing amyloidogenic processes. Here we report that the molecular tweezer CLR01 decreased aggregation and toxicity in induced pluripotent stem cell-derived dopaminergic cultures treated with PD brain protein extracts. In microfluidic devices CLR01 reduced alpha-synuclein aggregation in cell somas when axonal terminals were exposed to alpha-synuclein oligomers. We then tested CLR01 in vivo in a humanized alpha-synuclein overexpressing mouse model; mice treated at 12 months of age when motor defects are mild exhibited an improvement in motor defects and a decreased oligomeric alpha-synuclein burden. Finally, CLR01 reduced alpha-synuclein-associated pathology in mice injected with alpha-synuclein aggregates into the striatum or substantia nigra. Taken together, these results highlight CLR01 as a disease-modifying therapy for PD and support further clinical investigation.

"LRRK2: Autophagy and Lysosomal Activity".

It has been 15 years since the Leucine-rich repeat kinase 2 (LRRK2) gene was identified as the most common genetic cause for Parkinson's disease (PD). The two most common mutations are the LRRK2-G2019S, located in the kinase domain, and the LRRK2-R1441C, located in the ROC-COR domain. While the LRRK2-G2019S mutation is associated with increased kinase activity, the LRRK2-R1441C exhibits a decreased GTPase activity and altered kinase activity. Multiple lines of evidence have linked the LRRK2 protein with a role in the autophagy pathway and with lysosomal activity in neurons. Neurons rely heavily on autophagy to recycle proteins and process cellular waste due to their post-mitotic state. Additionally, lysosomal activity decreases with age which can potentiate the accumulation of α-synuclein, the pathological hallmark of PD, and subsequently lead to the build-up of Lewy bodies (LBs) observed in this disorder. This review provides an up to date summary of the LRRK2 field to understand its physiological role in the autophagy pathway in neurons and related cells. Careful assessment of how LRRK2 participates in the regulation of phagophore and autophagosome formation, autophagosome and lysosome fusion, lysosomal maturation, maintenance of lysosomal pH and calcium levels, and lysosomal protein degradation are addressed. The autophagy pathway is a complex cellular process and due to the variety of LRRK2 models studied in the field, associated phenotypes have been reported to be seemingly conflicting. This review provides an in-depth discussion of different models to assess the normal and disease-associated role of the LRRK2 protein on autophagic function. Given the importance of the autophagy pathway in Parkinson's pathogenesis it is particularly relevant to focus on the role of LRRK2 to discover novel therapeutic approaches that restore lysosomal protein degradation homeostasis.

Recommendations for measuring whisker movements and locomotion in mice with sensory, motor and cognitive deficits.

BACKGROUND: Previous studies have measured whisker movements and locomotion to characterise mouse models of neurodegenerative disease. However, these studies have always been completed in isolation, and do not involve standardized procedures for comparisons across multiple mouse models and background strains. NEW METHOD: We present a standard method for conducting whisker movement and locomotion studies, by carrying out qualitative scoring and quantitative measurement of whisker movements from high-speed video footage of mouse models of Amyotrophic Lateral Sclerosis, Huntington's disease, Parkinson's disease, Alzheimer's disease, Cerebellar Ataxia, Somatosensory Cortex Development and Ischemic stroke. RESULTS: Sex, background strain, source breeder and genotype all affected whisker movements. All mouse models, apart from Parkinson's disease, revealed differences in whisker movements during locomotion. R6/2 CAG250 Huntington's disease mice had the strongest behavioural phenotype. Robo3R3-5-CKO and RIM-DKOSert mouse models have abnormal somatosensory cortex development and revealed significant changes in whisker movements during object exploration. COMPARISON WITH EXISTING METHOD(S): Our results have good agreement with past studies, which indicates the robustness and reliability of measuring whisking. We recommend that differences in whisker movements of mice with motor deficits can be captured in open field arenas, but that mice with impairments to sensory or cognitive functioning should also be filmed investigating objects. Scoring clips qualitatively before tracking will help to structure later analyses. CONCLUSIONS: Studying whisker movements provides a quantitative measure of sensing, motor control and exploration. However, the effect of background strain, sex and age on whisker movements needs to be better understood.

LRRK2 interacts with the vacuolar-type H+-ATPase pump a1 subunit to regulate lysosomal function.

Lysosomal dysfunction lies at the centre of the cellular mechanisms underlying Parkinson's disease although the precise underlying mechanisms remain unknown. We investigated the role of leucine-rich repeat kinase 2 (LRRK2) on lysosome biology and the autophagy pathway in primary neurons expressing the human LRRK2-G2019S or LRKK2-R1441C mutant or the human wild-type (hWT-LRRK2) genomic locus. The expression of LRRK2-G2019S or hWT-LRRK2 inhibited autophagosome production, whereas LRRK2-R1441C induced a decrease in autophagosome/lysosome fusion and increased lysosomal pH. In vivo data from the cortex and substantia nigra pars compacta of aged LRRK2 transgenic animals revealed alterations in autophagosome puncta number reflecting those phenotypes seen in vitro. Using the two selective and potent LRRK2 kinase inhibitors, MLi-2 and PF-06447475, we demonstrated that the LRRK2-R1441C-mediated decrease in autolysosome maturation is not dependent on LRRK2 kinase activity. We showed that hWT-LRRK2 and LRRK2-G2019S bind to the a1 subunit of vATPase, which is abolished by the LRRK2-R1441C mutation, leading to a decrease in a1 protein and cellular mislocalization. Modulation of lysosomal zinc increased vATPase a1 protein levels and rescued the LRRK2-R1441C-mediated cellular phenotypes. Our work defines a novel interaction between the LRRK2 protein and the vATPase a1 subunit and demonstrates a mode of action by which drugs may rescue lysosomal dysfunction. These results demonstrate the importance of LRRK2 in lysosomal biology, as well as the critical role of the lysosome in PD.

RNA sequencing reveals MMP2 and TGFB1 downregulation in LRRK2 G2019S Parkinson's iPSC-derived astrocytes.

Non-neuronal cell types such as astrocytes can contribute to Parkinson's disease (PD) pathology. The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is one of the most common known causes of familial PD. To characterize its effect on astrocytes, we developed a protocol to produce midbrain-patterned astrocytes from human induced pluripotent stem cells (iPSCs) derived from PD LRRK2 G2019S patients and healthy controls. RNA sequencing analysis revealed the downregulation of genes involved in the extracellular matrix in PD cases. In particular, transforming growth factor beta 1 (TGFB1), which has been shown to inhibit microglial inflammatory response in a rat model of PD, and matrix metallopeptidase 2 (MMP2), which has been shown to degrade α-synuclein aggregates, were found to be down-regulated in LRRK2 G2019S astrocytes. Our findings suggest that midbrain astrocytes carrying the LRRK2 G2019S mutation may have reduced neuroprotective capacity and may contribute to the development of PD pathology.

An integrated transcriptomics and proteomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2.

BACKGROUND: Mutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease, and the relevance of LRRK2 to the sporadic form of the disease is becoming ever more apparent. It is therefore essential that studies are conducted to improve our understanding of the cellular role of this protein. Here we use multiple models and techniques to identify the pathways through which LRRK2 mutations may lead to the development of Parkinson's disease. METHODS: A novel integrated transcriptomics and proteomics approach was used to identify pathways that were significantly altered in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blotting, immunostaining and functional assays including FM1-43 analysis of synaptic vesicle endocytosis were performed to confirm these findings in iPSC-derived dopaminergic neuronal cultures carrying either the LRRK2-G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. RESULTS: Our integrated -omics analysis revealed highly significant dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blot analysis confirmed that key endocytic proteins including endophilin I-III, dynamin-1, and various RAB proteins were downregulated in these cultures and in cultures carrying the LRRK2-R1441C mutation, compared with controls. We also found changes in expression of 25 RAB proteins. Changes in endocytic protein expression led to a functional impairment in clathrin-mediated synaptic vesicle endocytosis. Further to this, we found that the endocytic pathway was also perturbed in striatal tissue of aged LRRK2 BAC transgenic rats overexpressing either the LRRK2 wildtype, LRRK2-R1441C or LRRK2-G2019S transgenes. Finally, we found that clathrin heavy chain and endophilin I-III levels are increased in human post-mortem tissue from LRRK2-G2019S patients compared with controls. CONCLUSIONS: Our study demonstrates extensive alterations across the endocytic pathway associated with LRRK2 mutations in iPSC-derived dopaminergic neurons and BAC transgenic rats, as well as in post-mortem brain tissue from PD patients carrying a LRRK2 mutation. In particular, we find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process.

Are rodent models of Parkinson's disease behaving as they should?

In recent years our understanding of Parkinson's disease has expanded both in terms of pathological hallmarks as well as relevant genetic influences. In parallel with the aetiological discoveries a multitude of PD animal models have been established. The vast majority of these are rodent models based on environmental, genetic and mechanistic insight. A major challenge in many of these models is their ability to only recapitulate some of the complex disease features seen in humans. Although symptom alleviation and clinical signs are of utmost importance in therapeutic research many of these models lack comprehensive behavioural testing. While non-motor symptoms become increasingly important as early diagnostic markers in PD, they are poorly characterized in rodents. In this review we look at well-established and more recent animal models of PD in terms of behavioural characterization and discuss how they can best contribute to progression in Parkinson's research.

Combinational losses of synucleins reveal their differential requirements for compensating age-dependent alterations in motor behavior and dopamine metabolism.

Synucleins are involved in multiple steps of the neurotransmitter turnover, but the largely normal synaptic function in young adult animals completely lacking synucleins suggests their roles are dispensable for execution of these processes. Instead, they may be utilized for boosting the efficiency of certain molecular mechanisms in presynaptic terminals, with a deficiency of synuclein proteins sensitizing to or exacerbating synaptic malfunction caused by accumulation of mild alterations, which are commonly associated with aging. Although functional redundancy within the family has been reported, it is unclear whether the remaining synucleins can fully compensate for the deficiency of a lost family member or whether some functions are specific for a particular member. We assessed several structural and functional characteristics of the nigrostriatal system of mice lacking members of the synuclein family in every possible combination and demonstrated that stabilization of the striatal dopamine level depends on the presence of α-synuclein and cannot be compensated by other family members, whereas ß-synuclein is required for efficient maintenance of animal's balance and coordination in old age.

LRRK2 BAC transgenic rats develop progressive, L-DOPA-responsive motor impairment, and deficits in dopamine circuit function.

Mutations in leucine-rich repeat kinase 2 (LRRK2) lead to late-onset, autosomal dominant Parkinson's disease, characterized by the degeneration of dopamine neurons of the substantia nigra pars compacta, a deficit in dopamine neurotransmission and the development of motor and non-motor symptoms. The most prevalent Parkinson's disease LRRK2 mutations are located in the kinase (G2019S) and GTPase (R1441C) encoding domains of LRRK2. To better understand the sequence of events that lead to progressive neurophysiological deficits in vulnerable neurons and circuits in Parkinson's disease, we have generated LRRK2 bacterial artificial chromosome transgenic rats expressing either G2019S or R1441C mutant, or wild-type LRRK2, from the complete human LRRK2 genomic locus, including endogenous promoter and regulatory regions. Aged (18-21 months) G2019S and R1441C mutant transgenic rats exhibit L-DOPA-responsive motor dysfunction, impaired striatal dopamine release as determined by fast-scan cyclic voltammetry, and cognitive deficits. In addition, in vivo recordings of identified substantia nigra pars compacta dopamine neurons in R1441C LRRK2 transgenic rats reveal an age-dependent reduction in burst firing, which likely results in further reductions to striatal dopamine release. These alterations to dopamine circuit function occur in the absence of neurodegeneration or abnormal protein accumulation within the substantia nigra pars compacta, suggesting that nigrostriatal dopamine dysfunction precedes detectable protein aggregation and cell death in the development of Parkinson's disease. In conclusion, our longitudinal deep-phenotyping provides novel insights into how the genetic burden arising from human mutant LRRK2 manifests as early pathophysiological changes to dopamine circuit function and highlights a potential model for testing Parkinson's therapeutics.

Forced cell cycle exit and modulation of GABAA, CREB, and GSK3ß signaling promote functional maturation of induced pluripotent stem cell-derived neurons.

Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na(+) current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABAAreceptor phenotype that was demonstrated by Ca(2+) imaging and the ability of GABAAreceptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.

A novel resource for studying function and dysfunction of α-synuclein: mouse lines for modulation of endogenous Snca gene expression.

Pathological modification of α-synuclein is believed to be an important event in pathogenesis of Parkinson's disease and several other neurodegenerative diseases. In normal cells this protein has been linked to many intracellular processes and pathways. However, neither normal function of α-synuclein in neuronal and certain other types of cells nor its exact role in the disease pathogenesis is well understood, which is largely due to limitations of animal models used for studying this protein. We produced and validated several novel mouse lines for manipulating expression of the endogenous Snca gene coding for α-synuclein. These include a line for conditional Cre-recombinase-driven inactivation of the gene; a line for conditional Flp-driven restoration of a neo-cassete-blocked α-synuclein expression; a new line with a "clean" constituent knockout of the gene as well as a line carrying this knockout locus and Rosa26-stop-lacZ reporter locus linked at the same mouse chromosome 6. Altogether these lines represent a set of new useful tools for studies of α-synuclein normal function and the role of this protein in disease pathogenesis.