Vivek K. Unni

Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I

We have generated mice lacking synaptogyrin I and synaptophysin I to explore the functions of these abundant tyrosine-phosphorylated proteins of synaptic vesicles. Single and double knockout mice were alive and fertile without significant morphological or biochemical changes. Electrophysiological recordings in the hippocampal CA1 region revealed that short-term and long-term synaptic plasticity were severely reduced in the synaptophysin/synaptogyrin double knockout mice. LTP was decreased independent of the induction protocol, suggesting that the defect in LTP was not caused by insufficient induction. Our data show that synaptogyrin I and synaptophysin I perform redundant and essential functions in synaptic plasticity without being required for neurotransmitter release itself.

Distinct Roles In Vivo for the Ubiquitin–Proteasome System and the Autophagy–Lysosomal Pathway in the Degradation of α-Synuclein

Increased intracellular levels of α-synuclein are implicated in Parkinsons disease and related disorders and may be caused by alterations in the ubiquitin–proteasome system (UPS) or the autophagy–lysosomal pathway (ALP). A critical question remains how α-synuclein is degraded by neurons in vivo. To address this, our study uses α-synuclein transgenic mice, expressing human α-synuclein or α-synuclein-eGFP under the (h)PDGF-β promoter, in combination with in vivo pharmacologic and multiphoton imaging strategies to systematically test degradation pathways in the living mouse brain. We demonstrate that the UPS is the main degradation pathway for α-synuclein under normal conditions in vivo while with increased α-synuclein burden the ALP is recruited. Moreover, we report alterations of the UPS in α-synuclein transgenic mice and age dependence to the role of the UPS in α-synuclein degradation. In addition, we provide evidence that the UPS and ALP might be functionally connected such that impairment of one can upregulate the other. These results provide a novel link between the UPS, the ALP, and α-synuclein pathology and may have important implications for future therapeutics targeting degradation pathways.

NETWORKS OF COACTIVE NEURONS IN DEVELOPING LAYER 1

Spontaneous neuronal activity plays an important role in the development of cortical circuitry, yet its spatio-temporal dynamics are poorly understood. Cajal-Retzius (CR) neurons in developing layer 1 are necessary for correct cortical lamination and are strategically located to coordinate early circuit activity. To characterize the spontaneous activity of CR and other layer 1 neurons during cortical development, we imaged calcium transients in populations of layer 1 neurons in hemispheres and slices from postnatal rat somato-sensory neocortex. The spontaneous activity in layer 1 had complex spatio-temporal patterns. Groups of non-CR cells showed synchronous activations and formed networks of correlated neurons superimposed in the same territory. Correlated activity among non-CR cells was mediated by a depolarizing effect of GABA and was modulated by glutamate, probably released by CR cells. Our findings demonstrate that developing layer 1 can sustain complex patterns of correlated activity and reveal a circuit mechanism that can mediate this patterned activity.

Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export

Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC.

Progressive aggregation of alpha-synuclein and selective degeneration of lewy inclusion-bearing neurons in a mouse model of parkinsonism.

Summary Aggregated alpha-synuclein inclusions are found where cell death occurs in several diseases, including Parkinson’s Disease, Dementia with Lewy Bodies and Multiple System Atrophy. However, the relationship between inclusion formation and an individual cell’s fate has been difficult to study with conventional techniques. We developed a system that allows for in vivo imaging of the same neurons over months. We show that intracerebral injection of preformed fibrils of recombinant alpha-synuclein can seed aggregation of transgenically-expressed and endogenous alpha-synuclein in neurons. Somatic inclusions undergo a stage-like maturation, with progressive compaction coinciding with decreased soluble somatic and nuclear alpha-synuclein. Mature inclusions bear the post-translational hallmarks of human Lewy pathology. Long-term imaging of inclusion-bearing neurons and neighboring neurons without inclusions demonstrates selective degeneration of inclusion-bearing cells. Our results indicate that inclusion formation is tightly correlated with cellular toxicity and that seeding may be a pathologically relevant mechanism of progressive neurodegeneration in many synucleinopathies.

Calcium Release from Presynaptic Ryanodine-Sensitive Stores Is Required for Long-Term Depression at Hippocampal CA3-CA3 Pyramidal Neuron Synapses

Although Ca2+ release from internal stores has been proposed to be important for the induction of long-term synaptic plasticity, the importance of Ca2+ stores localized in presynaptic terminals remains unclear. Here, we have selectively applied pharmacological antagonists to either the presynaptic or postsynaptic cell in paired whole-cell recordings from hippocampal CA3 pyramidal neurons in slice culture. We demonstrate directly the necessary role of presynaptic, but not postsynaptic, ryanodine-sensitive Ca2+ stores in the induction of NMDA receptor (NMDAR)-dependent long-term depression (LTD). Using two-photon laser scanning microscopy, we further find that release from the ryanodine-sensitive stores during prolonged synaptic stimulation generates a slowly rising Ca2+ signal in the presynaptic terminal that is required for the induction of LTD. Moreover, this form of LTD has a significant presynaptic component of expression because it causes a marked decrease in the rate of release from CA3 neuron presynaptic terminals of FM 1-43, a fluorescent probe of synaptic vesicle cycling. Thus, Ca2+ release from presynaptic ryanodine-sensitive stores is critical in the induction of a presynaptic component of NMDAR-dependent LTD.

Presynaptic Alpha-Synuclein Aggregation in a Mouse Model of Parkinson's Disease

Parkinsons disease and dementia with Lewy bodies are associated with abnormal neuronal aggregation of α-synuclein. However, the mechanisms of aggregation and their relationship to disease are poorly understood. We developed an in vivo multiphoton imaging paradigm to study α-synuclein aggregation in mouse cortex with subcellular resolution. We used a green fluorescent protein-tagged human α-synuclein mouse line that has moderate overexpression levels mimicking human disease. Fluorescence recovery after photobleaching (FRAP) of labeled protein demonstrated that somatic α-synuclein existed primarily in an unbound, soluble pool. In contrast, α-synuclein in presynaptic terminals was in at least three different pools: (1) as unbound, soluble protein; (2) bound to presynaptic vesicles; and (3) as microaggregates. Serial imaging of microaggregates over 1 week demonstrated a heterogeneous population with differing α-synuclein exchange rates. The microaggregate species were resistant to proteinase K, phosphorylated at serine-129, oxidized, and associated with a decrease in the presynaptic vesicle protein synapsin and glutamate immunogold labeling. Multiphoton FRAP provided the specific binding constants for α-synucleins binding to synaptic vesicles and its effective diffusion coefficient in the soma and axon, setting the stage for future studies targeting synuclein modifications and their effects. Our in vivo results suggest that, under moderate overexpression conditions, α-synuclein aggregates are selectively found in presynaptic terminals.

Alpha-synuclein's degradation in vivo: Opening a new (cranial) window on the roles of degradation pathways in Parkinson disease

Progressive accumulation of α-synuclein is key to the pathology of many neurodegenerative diseases, including Parkinson disease and dementia with Lewy bodies. Increased intracellular levels of α-synuclein may be caused by enhanced expression or alterations in protein degradation pathways. Here we review our recent study showing that the ubiquitin-proteasome system and the autophagy-lysosomal pathway are differentially involved in α-synucleins degradation in vivo. We discuss the key findings obtained with our novel in vivo approach and also present a model for the progression of protein aggregation and dysfunctional degradation in Parkinson disease.

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

Pathologic inclusions define α-synucleinopathies that include Parkinsons 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 Parkinsons 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.

In Vivo Imaging of α-Synuclein in Mouse Cortex Demonstrates Stable Expression and Differential Subcellular Compartment Mobility

Background Regulation of α-synuclein levels within cells is thought to play a critical role in Parkinsons Disease (PD) pathogenesis and in other related synucleinopathies. These processes have been studied primarily in reduced preparations, including cell culture. We now develop methods to measure α-synuclein levels in the living mammalian brain to study in vivo protein mobility, turnover and degradation with subcellular specificity. Methodology/Principal Findings We have developed a system using enhanced Green Fluorescent Protein (GFP)-tagged human α-synuclein (Syn-GFP) transgenic mice and in vivo multiphoton imaging to measure α-synuclein levels with subcellular resolution. This new experimental paradigm allows individual Syn-GFP-expressing neurons and presynaptic terminals to be imaged in the living mouse brain over a period of months. We find that Syn-GFP is stably expressed by neurons and presynaptic terminals over this time frame and further find that different presynaptic terminals can express widely differing levels of Syn-GFP. Using the fluorescence recovery after photobleaching (FRAP) technique in vivo we provide evidence that at least two pools of Syn-GFP exist in terminals with lower levels of mobility than measured previously. These results demonstrate that multiphoton imaging in Syn-GFP mice is an excellent new strategy for exploring the biology of α-synuclein and related mechanisms of neurodegeneration. Conclusions/Significance In vivo multiphoton imaging in Syn-GFP transgenic mice demonstrates stable α-synuclein expression and differential subcellular compartment mobility within cortical neurons. This opens new avenues for studying α-synuclein biology in the living brain and testing new therapeutics for PD and related disorders.