Roles of neuronal lysosomes in the etiology of Parkinson's disease.

Therapeutic progress in neurodegenerative conditions such as Parkinson's disease has been hampered by a lack of detailed knowledge of its molecular etiology. The advancements in genetics and genomics have provided fundamental insights into specific protein players and the cellular processes involved in the onset of disease. In this respect, the autophagy-lysosome system has emerged in recent years as a strong point of convergence for genetics, genomics, and pathologic indications, spanning both familial and idiopathic Parkinson's disease. Most, if not all, genes linked to familial disease are involved, in a regulatory capacity, in lysosome function (e.g., LRRK2, alpha-synuclein, VPS35, Parkin, and PINK1). Moreover, the majority of genomic loci associated with increased risk of idiopathic Parkinson's cluster in lysosome biology and regulation (GBA as the prime example). Lastly, neuropathologic evidence showed alterations in lysosome markers in autoptic material that, coupled to the alpha-synuclein proteinopathy that defines the disease, strongly indicate an alteration in functionality. In this Brief Review article, I present a personal perspective on the molecular and cellular involvement of lysosome biology in Parkinson's pathogenesis, aiming at a larger vision on the events underlying the onset of the disease. The attempts at targeting autophagy for therapeutic purposes in Parkinson's have been mostly aimed at "indiscriminately" enhancing its activity to promote the degradation and elimination of aggregate protein accumulations, such as alpha-synuclein Lewy bodies. However, this approach is based on the assumption that protein pathology is the root cause of disease, while pre-pathology and pre-degeneration dysfunctions have been largely observed in clinical and pre-clinical settings. In addition, it has been reported that unspecific boosting of autophagy can be detrimental. Thus, it is important to understand the mechanisms of specific autophagy forms and, even more, the adjustment of specific lysosome functionalities. Indeed, lysosomes exert fine signaling capacities in addition to their catabolic roles and might participate in the regulation of neuronal and glial cell functions. Here, I discuss hypotheses on these possible mechanisms, their links with etiologic and risk factors for Parkinson's disease, and how they could be targeted for disease-modifying purposes.

The small GTPase Rit2 modulates LRRK2 kinase activity, is required for lysosomal function and protects against alpha-synuclein neuropathology.

In Parkinson's disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear, but they are hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, and LRRK2 kinase activity has been shown to be involved in pS129-aSyn inclusion modulation. We observed selective downregulation of the novel PD risk factor RIT2 in vitro and in vivo. Rit2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rit2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, Rit2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. On the other hand, reduction of Rit2 levels leads to defects in the ALP, similar to those induced by the G2019S-LRRK2 mutation. Our data indicate that Rit2 is required for correct lysosome function, inhibits overactive LRRK2 to ameliorate ALP impairment, and counteracts aSyn aggregation and related deficits. Targeting Rit2 could represent an effective strategy to combat neuropathology in familial and idiopathic PD.

Lysosomal Pathogenesis of Parkinson's Disease: Insights From LRRK2 and GBA1 Rodent Models.

The discovery of mutations in LRRK2 and GBA1 that are linked to Parkinson's disease provided further evidence that autophagy and lysosome pathways are likely implicated in the pathogenic process. Their protein products are important regulators of lysosome function. LRRK2 has kinase-dependent effects on lysosome activity, autophagic efficacy and lysosomal Ca2+ signaling. Glucocerebrosidase (encoded by GBA1) is a hydrolytic enzyme contained in the lysosomes and contributes to the degradation of alpha-synuclein. PD-related mutations in LRRK2 and GBA1 slow the degradation of alpha-synuclein, thus directly implicating the dysfunction of the process in the neuropathology of Parkinson's disease. The development of genetic rodent models of LRRK2 and GBA1 provided hopes of obtaining reliable preclinical models in which to study pathogenic processes and perform drug validation studies. Here, I will review the extensive characterization of these models, their impact on understanding lysosome alterations in the course of Parkinson's disease and what novel insights have been obtained. In addition, I will discuss how these models fare with respect to the features of a "gold standard" animal models and what could be attempted in future studies to exploit LRRK2 and GBA1 rodent models in the fight against Parkinson's disease.

Modeling Parkinson's disease in LRRK2 mice: focus on synaptic dysfunction and the autophagy-lysosomal pathway.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with familial and sporadic forms of Parkinson's disease (PD), for which the LRRK2 locus itself represents a risk factor. Idiopathic and LRRK2-related PD share the main clinical and neuropathological features, thus animals harboring the most common LRRK2 mutations, i.e. G2019S and R1441C/G, have been generated to replicate the parkinsonian phenotype and investigate the underlying pathological mechanisms. Most LRRK2 rodent models, however, fail to show the main neuropathological hallmarks of the disease i.e. the degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of Lewy bodies or Lewy body-like aggregates of α-synuclein, lacking face validity. Rather, they manifest dysregulation in cellular pathways and functions that confer susceptibility to a variety of parkinsonian toxins/triggers and model the presymptomatic/premotor stages of the disease. Among such susceptibility factors, dysregulation of synaptic activity and proteostasis are evident in LRRK2 mutants. These abnormalities are also manifest in the PD brain and represent key events in the development and progression of the pathology. The present minireview covers recent articles (2018-2021) investigating the role of LRRK2 and LRRK2 mutants in the regulation of synaptic activity and autophagy-lysosomal pathway. These articles confirm a perturbation of synaptic vesicle endocytosis and glutamate release in LRRK2 mutants. Likewise, LRRK2 mutants show a marked impairment of selective forms of autophagy (i.e. mitophagy and chaperone-mediated autophagy) and lysosomal function, with minimal perturbations of nonselective autophagy. Thus, LRRK2 rodents might help understand the contribution of these pathways to PD.

LRRK2 along the Golgi and lysosome connection: a jamming situation.

Parkinson's disease (PD) is an age-related neurodegenerative disorder, clinically characterized by bradykinesia, rigidity, and resting tremor. Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein containing two enzymatic domains. Missense mutations in its coding sequence are amongst the most common causes of familial PD. The physiological and pathological impact of LRRK2 is still obscure, but accumulating evidence supports a role for LRRK2 in membrane and vesicle trafficking, mainly functioning in the endosome-recycling system, (synaptic) vesicle trafficking, autophagy, and lysosome biology. LRRK2 binds and phosphorylates key regulators of the endomembrane systems and is dynamically localized at the Golgi. The impact of LRRK2 on the Golgi may reverberate throughout the entire endomembrane system and occur in multiple intersecting pathways, including endocytosis, autophagy, and lysosomal function. This would lead to overall dysregulation of cellular homeostasis and protein catabolism, leading to neuronal dysfunction and accumulation of toxic protein species, thus underlying the possible neurotoxic effect of LRRK2 mutations causing PD.

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.

Kinase inhibition of G2019S-LRRK2 enhances autolysosome formation and function to reduce endogenous alpha-synuclein intracellular inclusions.

The Parkinson's disease (PD)-associated kinase Leucine-Rich Repeat Kinase 2 (LRRK2) is a crucial modulator of the autophagy-lysosome pathway, but unclarity exists on the precise mechanics of its role and the direction of this modulation. In particular, LRRK2 is involved in the degradation of pathological alpha-synuclein, with pathogenic mutations precipitating neuropathology in cellular and animal models of PD, and a significant proportion of LRRK2 patients presenting Lewy neuropathology. Defects in autophagic processing and lysosomal degradation of alpha-synuclein have been postulated to underlie its accumulation and onset of neuropathology. Thus, it is critical to obtain a comprehensive knowledge on LRRK2-associated pathology. Here, we investigated a G2019S-LRRK2 recombinant cell line exhibiting accumulation of endogenous, phosphorylated alpha-synuclein. We found that G2019S-LRRK2 leads to accumulation of LC3 and abnormalities in lysosome morphology and proteolytic activity in a kinase-dependent fashion, but independent from constitutively active Rab10. Notably, LRRK2 inhibition was ineffective upon upstream blockade of autophagosome-lysosome fusion events, highlighting this step as critical for alpha-synuclein clearance.

Alpha-Synuclein and LRRK2 in Synaptic Autophagy: Linking Early Dysfunction to Late-Stage Pathology in Parkinson's Disease.

The lack of effective disease-modifying strategies is the major unmet clinical need in Parkinson´s disease. Several experimental approaches have attempted to validate cellular targets and processes. Of these, autophagy has received considerable attention in the last 20 years due to its involvement in the clearance of pathologic protein aggregates and maintenance of neuronal homeostasis. However, this strategy mainly addresses a very late stage of the disease, when neuropathology and neurodegeneration have likely "tipped over the edge" and disease modification is extremely difficult. Very recently, autophagy has been demonstrated to modulate synaptic activity, a process distinct from its catabolic function. Abnormalities in synaptic transmission are an early event in neurodegeneration with Leucine-Rich Repeat Kinase 2 (LRRK2) and alpha-synuclein strongly implicated. In this review, we analyzed these processes separately and then discussed the unification of these biomolecular fields with the aim of reconstructing a potential "molecular timeline" of disease onset and progression. We postulate that the elucidation of these pathogenic mechanisms will form a critical basis for the design of novel, effective disease-modifying therapies that could be applied early in the disease process.

Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons.

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.

A new hypothesis for Parkinson's disease pathogenesis: GTPase-p38 MAPK signaling and autophagy as convergence points of etiology and genomics.

The combination of genetics and genomics in Parkinson´s disease has recently begun to unveil molecular mechanisms possibly underlying disease onset and progression. In particular, catabolic processes such as autophagy have been increasingly gaining relevance as post-mortem evidence and experimental models suggested a participation in neurodegeneration and alpha-synuclein Lewy body pathology. In addition, familial Parkinson´s disease linked to LRRK2 and alpha-synuclein provided stronger correlation between etiology and alterations in autophagy. More detailed cellular pathways are proposed and genetic risk factors that associate with idiopathic Parkinson´s disease provide further clues in dissecting contributions of single players. Nevertheless, the fine-tuning of these processes remains elusive, as the initial stages of the pathways are not yet clarified.In this review, we collect literature evidence pointing to autophagy as the common, downstream target of Parkinsonian dysfunctions and augment current knowledge on the factors that direct the subsequent steps. Cell and molecular biology evidence indicate that p38 signaling underlies neurodegeneration and autoptic observations suggest a participation in neuropathology. Moreover, alpha-synuclein and LRRK2 also appear involved in the p38 pathway with additional roles in the regulation of GTPase signaling. Small GTPases are critical modulators of p38 activation and thus, their functional interaction with aSyn and LRRK2 could explain much of the detailed mechanics of autophagy in Parkinson´s disease.We propose a novel hypothesis for a more comprehensive working model where autophagy is controlled by upstream pathways, such as GTPase-p38, that have been so far underexplored in this context. In addition, etiological factors (LRRK2, alpha-synuclein) and risk loci might also combine in this common mechanism, providing a powerful experimental setting to dissect the cause of both familial and idiopathic disease.

Cryopreservation of Primary Mouse Neurons: The Benefit of Neurostore Cryoprotective Medium.

Primary neuronal culture from rodents is a well-established model to investigate cellular neurobiology in vitro. However, for this purpose cell cultures need to be generated expressly, requiring extensive animal handling. Furthermore, often the preparation of fresh culture generates an excess of cells that are ultimately wasted. Therefore the ability to successfully cryopreserve primary neural cells would represent an important resource for neuroscience research and would allow to significantly reduce the sacrifice of animals. We describe here a novel freezing medium that allows long-term cryopreservation of primary mouse neurons prepared from E15.5 embryos. Combining imaging, biochemical and electrophysiological analyses, we found that cryopreserved cultures are viable and mature regarding morphology and functionality. These findings suggest that cryopreserved neurons are a valuable alternative to acutely dissociated neural cultures.

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.

CADPS2 gene expression is oppositely regulated by LRRK2 and alpha-synuclein.

The Ca2+-dependent activator protein for secretion 2 (CADPS2) is a member of the CAPS/CADPS protein family that plays crucial roles in synaptic vesicle dynamics. Genomic variability in the CADPS2 gene has been associated to autism spectrum disorders and Alzheimer's disease, both characterized by altered neurotransmission. Biological evidence also linked CADPS2 to Parkinson's disease (PD), as a disease-causing mutation in leucine-rich repeat kinase 2 (LRRK2) was reported to increase CADPS2 gene and protein expression. Furthermore, restoration of CADPS2 physiologic levels was able to provide neuroprotection in patient-derived neurons, consistent with the synaptic dysfunction postulated to underlie PD. However, little is known about the influence of PD-related proteins on transcriptional regulation of critical synaptic genes such as CADPS2. Here we aimed at investigating the transcriptional effects of LRRK2 and alpha-synuclein (aSyn) on CADPS2 gene expression, using a combination of in silico analyses and cell biology techniques. First, we identified a predicted promoter in the human CADPS2 genomic sequence, which we then utilized in a luciferase-based gene reporter assay. This approach enabled us to disclose a differential effect of high levels of LRRK2 and aSyn on CADPS2 promoter activity. Specifically, CADPS2 transcriptional activity was enhanced by high cellular levels of LRRK2 and reduced by overexpression of aSyn. Consistently, CADPS2 mRNA levels were diminished in aSyn overexpressing cells. Our results indicate that LRRK2 and aSyn participate in the dysregulation of CADPS2 by altering transcription and support the hypothesis that synaptic dysfunctions, through different mechanisms, might contribute to the neuronal defects of diseases such as PD.

LRRK2 mouse models: dissecting the behavior, striatal neurochemistry and neurophysiology of PD pathogenesis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of familial Parkinson's disease (PD), resembling the sporadic disorder. Intensive effort has been directed toward LRRK2 mouse modeling and investigation, aimed at reproducing the human disease to inform mechanistic studies of pathogenesis and design of neuroprotective therapies. The physiological function of LRRK2 is still under exploration, but a clear role in striatal neurophysiology and animal behavior has emerged. Alterations in LRRK2 impair dopamine (DA) transmission, regulation and signaling, in addition to corticostriatal synaptic plasticity. Consistently, several subtle abnormalities in motor and nonmotor abilities have been demonstrated in LRRK2 genetic mouse models, generally paralleling preclinical symptoms of early DA dysfunction. However, the variability in model design and phenotypes observed requires a critical approach in interpreting the results, adapting the model used to the specific research question. Etiologically appropriate knockin mice might represent the ultimate animal model in which to study early disease mechanisms and therapies as well as to investigate drug effectiveness and off-target consequences.

Elevated levels of alpha-synuclein blunt cellular signal transduction downstream of Gq protein-coupled receptors.

Alpha-synuclein is central to Parkinson's disease pathogenesis and pathology, however its precise functions are still unclear. It has been shown to bind both PLCß1 and MAPKs, but how this property influences the downstream signaling of Gq protein-coupled receptors has not been elucidated. Here we show that recombinant expression of alpha-synuclein in human neuroblastoma cells enhances cellular levels of PLCß1 but blunts its signaling pathway, preventing the agonist-dependent rise of cytoplasmic Ca2+. In addition, overexpressing alpha-synuclein abolishes the activation of ERK1/2 upon agonist stimulation, indicating an upstream action in the signal transduction pathway. This data demonstrates that alpha-synuclein, when recombinantly expressed, interferes with the normal signaling of Gq-protein coupled receptors, which are then dysfunctional. Since many neurotransmitter systems utilize these receptor signaling pathways to mediate different abilities affected in Parkinson's disease, we argue this novel perspective might be helpful in designing treatment strategies for some of the non-motor symptoms in Parkinson's disease and synucleinopathies.

Insights from late-onset familial parkinsonism on the pathogenesis of idiopathic Parkinson's disease.

Disease-modifying therapies that slow or halt the progression of Parkinson's disease are an unmet clinical need. Many hypotheses have been put forward to explain the pathogenesis of the disease, but none has led to the development of disease-modifying drugs. Here we focus on familial forms of late-onset parkinsonism that most closely resemble idiopathic Parkinson's disease and present a synthesis of emerging molecular advances. Genetic discoveries and mechanistic investigations have highlighted early alterations to synaptic function, endosomal maturation, and protein sorting that might lead to an intracellular proteinopathy. We propose that these cellular processes constitute one pathway to pathogenesis and suggest that neuroprotection, as an adjunct to current symptomatic treatments, need not remain an elusive goal.

Chronic and acute LRRK2 silencing has no long-term behavioral effects, whereas wild-type and mutant LRRK2 overexpression induce motor and cognitive deficits and altered regulation of dopamine release.

INTRODUCTION: Germline silencing of the PD-related protein LRRK2 does not alter glutamate or dopamine release in adult mice, but some exploratory abnormalities have been reported with ageing. Contrastingly, high levels of human LRRK2 cause locomotor alterations and cognitive deficits accompanied by reduced striatal dopamine levels, with the latter also observed in G2019S mutant mice. Comparative cognitive and motor behavioral testing of LRRK2 KO, overexpressor and mutant overexpressor mice has not previously been reported. METHODS: Parallel, comparative behavioral characterization was performed assessing motor and cognitive abilities. Striatal antisense oligonucleotide injections were conducted to investigate the effects of acute LRRK2 silencing on behavior and dopamine fiber density. Striatal synaptosomes prepared from hG2019S mice assessed vesicular release of dopamine and its sensitivity to D2 autoreceptor stimulation. RESULTS: Genetic ablation of LRRK2 has no long-term consequences on motor or cognitive function. Consistently, no effects on behavior or dopaminergic fiber density were observed following acute striatal silencing. Conversely, 12-month OE mice show persistent locomotor deficits and worsening of cognitive abilities; whereas, hG2019S mice display early hyperactivity and effective learning and memory that progress to decreased motor and cognitive deficits at older ages. The G2019S mutation does not affect vesicular dopamine release, but decreases its sensitivity to D2-mediated inhibition. CONCLUSION: LRRK2 silencing is well tolerated in mouse, arguing PD does not result from LRRK2 loss of function. High levels of WT and G2019S LRRK2 produce similar but temporally distinct phenotypes, potentially modeling different stages of disease progression. The data implicate gain of LRRK2 function in the pathogenesis of PD.

LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory.

Mutations in leucine-rich repeat kinase 2 (Lrrk2) are the most common genetic cause of Parkinson's disease (PD), a neurodegenerative disorder affecting 1-2% of those >65 years old. The neurophysiology of LRRK2 remains largely elusive, although protein loss suggests a role in glutamatergic synapse transmission and overexpression studies show altered dopamine release in aged mice. We show that glutamate transmission is unaltered onto striatal projection neurons (SPNs) of adult LRRK2 knockout mice and that adult animals exhibit no detectable cognitive or motor deficits. Basal synaptic transmission is also unaltered in SPNs of LRRK2 overexpressing mice, but they do exhibit clear alterations to D2-receptor-mediated short-term synaptic plasticity, behavioral hypoactivity and impaired recognition memory. These phenomena are associated with decreased striatal dopamine tone and abnormal dopamine- and cAMP-regulated phosphoprotein 32 kDa signal integration. The data suggest that LRRK2 acts at the nexus of dopamine and glutamate signaling in the adult striatum, where it regulates dopamine levels, presynaptic glutamate release via D2-dependent synaptic plasticity and dopamine-receptor signal transduction.

Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice.

Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.