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.

Calcium dysregulation combined with mitochondrial failure and electrophysiological maturity converge in Parkinson's iPSC-dopamine neurons.

Parkinson's disease (PD) is characterized by a progressive deterioration of motor and cognitive functions. Although death of dopamine neurons is the hallmark pathology of PD, this is a late-stage disease process preceded by neuronal dysfunction. Here we describe early physiological perturbations in patient-derived induced pluripotent stem cell (iPSC)-dopamine neurons carrying the GBA-N370S mutation, a strong genetic risk factor for PD. GBA-N370S iPSC-dopamine neurons show an early and persistent calcium dysregulation notably at the mitochondria, followed by reduced mitochondrial membrane potential and oxygen consumption rate, indicating mitochondrial failure. With increased neuronal maturity, we observed decreased synaptic function in PD iPSC-dopamine neurons, consistent with the requirement for ATP and calcium to support the increase in electrophysiological activity over time. Our work demonstrates that calcium dyshomeostasis and mitochondrial failure impair the higher electrophysiological activity of mature neurons and may underlie the vulnerability of dopamine neurons in PD.

Hyperexcitability in young iPSC-derived C9ORF72 mutant motor neurons is associated with increased intracellular calcium release.

A large hexanucleotide repeat expansion in the C9ORF72 gene is the most prevalent cause of amyotrophic lateral sclerosis (ALS). To better understand neuronal dysfunction during ALS progression, we studied motor neuron (MN) cultures derived from iPSC lines generated from C9ORF72 (C9) expansion carriers and unaffected controls. C9 and control MN cultures showed comparable mRNA levels for MN markers SMI-32, HB9 and ISL1 and similar MN yields (> 50% TUJ1/SMI-32 double-positive MNs). Using whole-cell patch clamp we showed that C9-MNs have normal membrane capacitance, resistance and resting potential. However, immature (day 40) C9-MNs exhibited a hyperexcitable phenotype concurrent with increased release of calcium (Ca2+) from internal stores, but with no changes to NaV and KV currents. Interestingly, this was a transient phenotype. By day 47, maturing C9-MNs demonstrated normal electrophysiological activity, displaying only subtle alterations on mitochondrial Ca2+ release. Together, these findings suggest the potential importance of a developmental component to C9ORF72-related ALS.

From Synaptic Dysfunction to Neuroprotective Strategies in Genetic Parkinson's Disease: Lessons From LRRK2.

The pathogenesis of Parkinson's disease (PD) is thought to rely on a complex interaction between the patient's genetic background and a variety of largely unknown environmental factors. In this scenario, the investigation of the genetic bases underlying familial PD could unveil key molecular pathways to be targeted by new disease-modifying therapies, still currently unavailable. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are responsible for the majority of inherited familial PD cases and can also be found in sporadic PD, but the pathophysiological functions of LRRK2 have not yet been fully elucidated. Here, we will review the evidence obtained in transgenic LRRK2 experimental models, characterized by altered striatal synaptic transmission, mitochondrial dysfunction, and α-synuclein aggregation. Interestingly, the processes triggered by mutant LRRK2 might represent early pathological phenomena in the pathogenesis of PD, anticipating the typical neurodegenerative features characterizing the late phases of the disease. A comprehensive view of LRRK2 neuronal pathophysiology will support the possible clinical application of pharmacological compounds targeting this protein, with potential therapeutic implications for patients suffering from both familial and sporadic PD.

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.

Atypical progressive supranuclear palsy with corticospinal tract degeneration.

Progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), sporadic multisystem tauopathy, and some forms of frontotemporal dementia with Parkinsonism linked to chromosome 17 are characterized by neuronal and glial lesions accumulating tau protein containing 4 conserved repeats in microtubule-binding domain (4R tau). Corticospinal tract degeneration is not a common feature of 4R tauopathies. Our objective was to describe 12 cases with pathologic features similar to those of PSP but with prominent corticospinal tract degeneration. We reviewed the historical records and neuropathologic evaluation using standardized sampling, immunohistochemistry, semiquantitative analysis, image analysis, and electron microscopy. The mean age at onset and illness duration was 71 and 5.7 years, respectively. Eight cases were female. Eleven cases had clinical evidence of prominent upper motor neuron disease plus extrapyramidal features. There was focal parasagittal cortical atrophy involving motor cortex and degeneration of corticospinal tract with sparing of lower motor neurons like in primary lateral sclerosis. Prominent tau pathology was found in oligodendrocytes in motor cortex, subjacent white matter, and corticospinal tract characterized by globular cytoplasmic filamentous inclusions that were immunoreactive for 4R tau. The clinicopathologic features of these 12 cases expand the spectrum of 4R tauopathies.

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.