Dayne Beccano-Kelly

DNAJC13 mutations in Parkinson disease

A Saskatchewan multi-incident family was clinically characterized with Parkinson disease (PD) and Lewy body pathology. PD segregates as an autosomal-dominant trait, which could not be ascribed to any known mutation. DNA from three affected members was subjected to exome sequencing. Genome alignment, variant annotation and comparative analyses were used to identify shared coding mutations. Sanger sequencing was performed within the extended family and ethnically matched controls. Subsequent genotyping was performed in a multi-ethnic case-control series consisting of 2928 patients and 2676 control subjects from Canada, Norway, Taiwan, Tunisia, and the USA. A novel mutation in receptor-mediated endocytosis 8/RME-8 (DNAJC13 p.Asn855Ser) was found to segregate with disease. Screening of cases and controls identified four additional patients with the mutation, of which two had familial parkinsonism. All carriers shared an ancestral DNAJC13 p.Asn855Ser haplotype and claimed Dutch-German-Russian Mennonite heritage. DNAJC13 regulates the dynamics of clathrin coats on early endosomes. Cellular analysis shows that the mutation confers a toxic gain-of-function and impairs endosomal transport. DNAJC13 immunoreactivity was also noted within Lewy body inclusions. In late-onset disease which is most reminiscent of idiopathic PD subtle deficits in endosomal receptor-sorting/recycling are highlighted by the discovery of pathogenic mutations VPS35, LRRK2 and now DNAJC13. With this latest discovery, and from a neuronal perspective, a temporal and functional ecology is emerging that connects synaptic exo- and endocytosis, vesicular trafficking, endosomal recycling and the endo-lysosomal degradative pathway. Molecular deficits in these processes are genetically linked to the phenotypic spectrum of parkinsonism associated with Lewy body pathology.

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.

Robotic multiwell planar patch-clamp for native and primary mammalian cells

Robotic multiwell planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. It has not, however, been adopted significantly in other important areas of ion channel research, where conventional patch-clamp remains the favored method. Here, we show the wider potential of the multiwell approach with the ability for efficient intracellular solution exchange, describing protocols and success rates for recording from a range of native and primary mammalian cells derived from blood vessels, arthritic joints and the immune and central nervous systems. The protocol involves preparing a suspension of single cells to be dispensed robotically into 4–8 microfluidic chambers each containing a glass chip with a small aperture. Under automated control, giga-seals and whole-cell access are achieved followed by preprogrammed routines of voltage paradigms and fast extracellular or intracellular solution exchange. Recording from 48 chambers usually takes 1–6 h depending on the experimental design and yields 16–33 cell recordings.

Leptin prevents hippocampal synaptic disruption and neuronal cell death induced by amyloid β

Accumulation of amyloid-β (Aβ) is a key event mediating the cognitive deficits in Alzheimers disease (AD) as Aβ promotes synaptic dysfunction and triggers neuronal death. Recent evidence has linked the hormone leptin to AD as leptin levels are markedly attenuated in AD patients. Leptin is also a potential cognitive enhancer as it facilitates the cellular events underlying hippocampal learning and memory. Here we show that leptin prevents the detrimental effects of Aβ(1-42) on hippocampal long-term potentiation. Moreover leptin inhibits Aβ(1-42)-driven facilitation of long-term depression and internalization of the 2-amino-3-(5-methyl-3-oxo-1,2- oxazol-4-yl)propanoic acid (AMPA) receptor subunit, GluR1, via activation of PI3-kinase. Leptin also protects cortical neurons from Aβ(1-42)-induced cell death by a signal transducer and activator of transcription-3 (STAT-3)-dependent mechanism. Furthermore, leptin inhibits Aβ(1-42)-mediated upregulation of endophilin I and phosphorylated tau in vitro, whereas cortical levels of endophilin I and phosphorylated tau are enhanced in leptin-insensitive Zucker fa/fa rats. Thus leptin benefits the functional characteristics and viability of neurons that degenerate in AD. These novel findings establish that the leptin system is an important therapeutic target in neurodegenerative conditions.

Retromer-dependent neurotransmitter receptor trafficking to synapses is altered by the Parkinson's disease VPS35 mutation p.D620N

Vacuolar protein sorting 35 (VPS35) is a core component of the retromer complex, crucial to endosomal protein sorting and intracellular trafficking. We recently linked a mutation in VPS35 (p.D620N) to familial parkinsonism. Here, we characterize human VPS35 and retromer function in mature murine neuronal cultures and investigate neuron-specific consequences of the p.D620N mutation. We find VPS35 localizes to dendritic spines and is involved in the trafficking of excitatory AMPA-type glutamate receptors (AMPARs). Fundamental neuronal processes, including excitatory synaptic transmission, AMPAR surface expression and synaptic recycling are altered by VPS35 overexpression. VPS35 p.D620N acts as a loss-of-function mutation with respect to VPS35 activity regulating synaptic transmission and AMPAR recycling in mouse cortical neurons and dopamine neuron-like cells produced from induced pluripotent stem cells of human p.D620N carriers. Such perturbations to synaptic function likely produce chronic pathophysiological stress upon neuronal circuits that may contribute to neurodegeneration in this, and other, forms of parkinsonism.

Palmitoylation of δ-catenin by DHHC5 mediates activity-induced synapse plasticity

Synaptic cadherin adhesion complexes are known to be key regulators of synapse plasticity. However, the molecular mechanisms that coordinate activity-induced modifications in cadherin localization and adhesion and the subsequent changes in synapse morphology and efficacy remain unknown. We demonstrate that the intracellular cadherin binding protein δ-catenin is transiently palmitoylated by DHHC5 after enhanced synaptic activity and that palmitoylation increases δ-catenin–cadherin interactions at synapses. Both the palmitoylation of δ-catenin and its binding to cadherin are required for activity-induced stabilization of N-cadherin at synapses and the enlargement of postsynaptic spines, as well as the insertion of GluA1 and GluA2 subunits into the synaptic membrane and the concomitant increase in miniature excitatory postsynaptic current amplitude. Notably, context-dependent fear conditioning in mice resulted in increased δ-catenin palmitoylation, as well as increased δ-catenin–cadherin associations at hippocampal synapses. Together these findings suggest a role for palmitoylated δ-catenin in coordinating activity-dependent changes in synaptic adhesion molecules, synapse structure and receptor localization that are involved in memory formation.

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 Parkinsons 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 Parkinsons disease.

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 Parkinsons 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.

Leptin: A Novel Therapeutic Target in Alzheimer's Disease?

It is well established that the hormone leptin circulates in the plasma in amounts proportional to body fat content and it regulates food intake and body weight via its actions in the hypothalamus. However, numerous studies have shown that leptin receptors are widely expressed throughout the CNS and evidence is growing that leptin plays a role in modulating a variety of neuronal processes. In particular, recent studies have highlighted a potential cognitive enhancing role for leptin as it regulates diverse aspects of hippocampal synaptic function that are thought to underlie learning and memory processes including glutamate receptor trafficking, dendritic morphology, and activity-dependent synaptic plasticity. Characterisation of the novel actions of leptin in limbic brain regions is providing valuable insights into leptins role in higher cognitive functions in health and disease.

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.