Paul E. Gallant

Leucine-Rich Repeat Kinase 2 Regulates the Progression of Neuropathology Induced by Parkinson’s Disease-related Mutant α-synuclein

Mutations in alpha-synuclein and Leucine-rich repeat kinase 2 (LRRK2) are linked to autosomal dominant forms of Parkinsons disease (PD). However, little is known about any potential pathophysiological interplay between these two PD-related genes. Here we show in transgenic mice that although overexpression of LRRK2 alone did not cause neurodegeneration, the presence of excess LRRK2 greatly accelerated the progression of neuropathological abnormalities developed in PD-related A53T alpha-synuclein transgenic mice. Moreover, we found that LRRK2 promoted the abnormal aggregation and somatic accumulation of alpha-synuclein in A53T mice, which likely resulted from the impairment of microtubule dynamics, Golgi organization, and the ubiquitin-proteasome pathway. Conversely, genetic ablation of LRRK2 preserved the Golgi structure and suppressed the aggregation and somatic accumulation of alpha-synuclein, and thereby delayed the progression of neuropathology in A53T mice. These findings demonstrate that overexpression of LRRK2 enhances alpha-synuclein-mediated cytotoxicity and suggest inhibition of LRRK2 expression as a potential therapeutic option for ameliorating alpha-synuclein-induced neurodegeneration.

Regulation of Hermissenda K+ Channels by Cytoplasmic and Membrane-Associated C-Kinase

Abstract: Pharmacologic activation of endogenous protein kinase C (PKC) together with elevation of the intracellular Ca2+ level was previously shown to cause reduction of two voltage‐dependent K+ currents (IA and Ica2+‐K+) across the soma membrane of the type B photoreceptor within the eye of the mollusc Hermissenda crassicornis. Similar effects were also found to persist for days after acquisition of a classically conditioned response. Also, the state of phosphorylation of a low‐molecular‐weight protein was changed only within the eyes of conditioned Hermissenda. To examine the role of PKC in causing K+ current changes as well as changes of phosphorylation during conditioning (and possibly other physiologic contexts), we studied here the effects of endogenous PKC activation and exogenous PKC injection on phosphorylation and K+ channel function. Several phosphoproteins (20, 25, 56, and 165 kilodaltons) showed differences in phosphorylation in response to PKC activators applied to intact nervous systems or to isolated eyes. Specific differences were observed for membrane and cytosolic fractions in response to both the phorbol ester 12‐deoxyphorbol 13‐isobutyrate 20‐acetate (DPBA) or exogenous PKC in the presence of Ca2+ and phosphatidylserine/diacylglycerol. Type B cells pretreated with DPBA responded to PKC injection with a persistent reduction of K+ currents. In the absence of DPBA, PKC injection also caused K+ current reduction only following Ca2+ loading conditions. However, the direct effect of PKC injection in the absence of DPBA was only to increase ICa2+_K+. According to a proposed model, the amplitude of the K+ currents would depend on the steady‐state balance of effects mediated by PKC within the cytoplasm and membrane‐associated PKC. The model further specifies that the effects on K+ currents of cytoplasmic PKC require an intervening proteolytic step. Such a model predicts that increasing the concentration of cytoplasmic protease, e.g., with trypsin, will increase K+ currents, whereas blocking endogenous protease, e.g., with leupeptin, will decrease K+ currents. These effects should be opposed by preexposure of the cells to DPBA. Furthermore, prior injection of leupeptin should block or reverse the effects of subsequent injection of PKC into the type B cell. All of these predictions were confirmed by results reported here. Taken together, the results of this and previous studies suggest that PKC regulation of membrane excitability critically depends on its cellular locus. The implications of such function for long‐term physiologic transformations are discussed.

Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain.

We recently showed by electron microscopy that the postsynaptic density (PSD) from hippocampal cultures undergoes rapid structural changes after ischemia‐like conditions. Here we report that similar structural changes occur after delay in transcardial perfusion fixation of the mouse brain. Delay in perfusion fixation, a condition that mimics ischemic stress, resulted in 70%, 90%, and 23% increases in the thickness of PSDs from the hippocampus (CA1), cerebral cortex (layer III), and cerebellar cortex (Purkinje spines), respectively. In step with PSD thickening, the amount of PSD‐associated α‐calcium calmodulin‐dependent protein kinase II (α‐ CaMKII) label increased more in cerebral cortical spines than in Purkinje spines. Although the Purkinje PSDs thickened only slightly after delayed fixation, they became highly curved, and many formed sub‐PSD spheres ∼80 nm in diameter that labeled for CaMKII. Delayed perfusion fixation also produced more cytoplamic CaMKII clusters (∼110 nm in diameter) in the somas of pyramidal cells (from hippocampus and cerebral cortex) than in Purkinje cells. Thus a short delay in perfusion fixation produces cell‐specific structural changes at PSDs and neuronal somas. Purkinje cells respond somewhat differently to delayed perfusion fixation, perhaps owing to their lower levels of CaMKII, and CaMKII binding proteins at PSDs. We present here a catalogue of structural changes that signal a perfusion fixation delay, thereby providing criteria by which to assess perfusion fixation quality in experimental structural studies of brain and to shed light on the subtle changes that occur in intact brain following metabolic stress. J. Comp. Neurol. 501:731–740, 2007.

Activity induced changes in the distribution of Shanks at hippocampal synapses

Dendritic spines contain a family of abundant scaffolding proteins known as Shanks, but little is known about how their distributions might change during synaptic activity. Here, pre-embedding immunogold electron microscopy is used to localize Shanks in synapses from cultured hippocampal neurons. We find that Shanks are preferentially located at postsynaptic densities (PSDs) as well as in a filamentous network near the PSD, extending up to 120 nm from the postsynaptic membrane. Application of sub-type specific antibodies shows that Shank2 is typically concentrated at and near PSDs while Shank1 is, in addition, distributed throughout the spine head. Depolarization with high K+ for 2 min causes transient, reversible translocation of Shanks towards the PSD that is dependent on extracellular Ca2+. The amount of activity-induced redistribution and subsequent recovery is pronounced for Shank1 but less so for Shank2. Thus, Shank1 appears to be a dynamic element within the spine, whose translocation could be involved in activity-induced, transient structural changes, while Shank2 appears to be a more stable element positioned at the interface of the PSD with the spine cytoplasm.

Calcium-induced proteolysis of spectrin and band 3 protein in rat erythrocyte membranes

Calcium-dependent protease activity capable of degrading a number of endogenous proteins was found in rat red blood cell membranes. This protease activity, like that found in human red blood cells, was activated by low concentrations of calcium, but in the rat red blood cells, unlike the human red blood cells, calcium-activated protease activity was membrane-bound. A number of endogenous membrane-bound proteins were degraded after the addition of calcium to the membranes. These included spectrin bands 1 and 2 as well as bands 3, 2.1, and 2.2. No calcium-induced aggregation (transglutaminase activity) was noted in the rat red blood cell membranes.

Axonal transport of tubulin and actin

Axonal transport is responsible for supplying the axonal processes with proteins that are synthesized in the cell body. Among the proteins that are moved by this mechanism are tubulin and actin, two major components of the cytoskeleton. Observation of the movement of metabolically labeled tubulin and actin in-vivo has demonstrated that tubulin and actin transport are reduced in various diseases and with age, but transport is increased during axonal growth and regeneration. These metabolic studies have also raised questions about the underlying mechanisms of slow axonal transport such as: what is the polymerization state of tubulin and actin during transport, what motors and tracks are responsible for their movement down the axon, and how are the transport motors coupled to tubulin and actin during transport? Since experiments using metabolically labeled tubulin and actin have not effectively addressed these questions, a variety of new in-vitro fluorescent microscopy techniques have been devised to investigate these questions. These fluorescent microscopy experiments have suggested that tubulin can be transported in the unpolymerized soluble state and that such transport of soluble tubulin relies on the presence of formed microtubule tracks. It is not yet known what motor or motors are responsible for tubulin or actin transport in axons or how such a motor(s) might be coupled to such an abundant soluble cargo.

The Direct Effects of Graded Axonal Compression on Axoplasm and Fast Axoplasmic Transport

Abstract. The direct effects of mechanical compression on axoplasm and fast axoplasmic transport were studied by videoenhanced differential interference microscopy. Single axons, isolated from the squid, were compressed with 0.5, 5, 20, or 100 gram (g) weights placed over a 1 millimeter (mm) length of axon. Brief compressions (10 seconds) at low pressures (0.5 g/mm) momentarily deformed the axon, but the axoplasm and axon returned to their normal shape and position after the pressure was removed, and no residual changes in axoplasmic structures, fast axoplasmic transport or membrane function were seen. Compressing the axon with 5–20 g/mm, however, broke the axoplasm at the site of the crush and squeezed the axoplasm out from under the compression site. Though the axoplasm usually returned to the crush site after the weight was removed and organelles continued to move in the axoplasm under the crush, the organelles failed to cross a dense line that marked the site of the rejoined axoplasm, instead they accumulated over time at the crush site. This result suggests that the blockage of fast transport at moderate compressions was due to a mechanical breakage of the axoplasm at the compression site. The plasma membrane was apparently not transected after moderate compressions (5–20 g/mm) since the resting membrane potential returned to nearly control levels after the weight was removed. Compressions with 100 g/mm, however, did break the plasma membrane as evidenced by the rapid and irreversible loss of the action potential and resting potential and the ion-dependent liquefaction of axoplasm and loss of all organelle transport at the 100 g/mm compression site. Thus, small mechanical pressures elastically deformed the axoplasm, moderate pressures mechanically broke the axoplasm, and high pressures broke the axoplasm and the plasma membrane.

RAPID TURNOVER OF SPINULES AT SYNAPTIC TERMINALS

Spinules found in brain consist of small invaginations of plasma membranes which enclose membrane evaginations from adjacent cells. Here, we focus on the dynamic properties of the most common type, synaptic spinules, which reside in synaptic terminals. In order to test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures (7-day-old rats, 10-14 days in culture) were exposed to high K+ for 0.5-5 min, and examined by electron microscopy. Virtually no synaptic spinules were found in control slices representing a basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K+. Spinule formation peaked with approximately 1 min treatment at 37 degrees C, decreased with prolonged treatment, and disappeared after 1-2 min of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4 degrees C. N-methyl-D-aspartic acid (NMDA) treatment also induced synaptic spinule formation, but to a lesser extent than high K+ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4 degrees C, extremely rare in slices allowed to recover at 28 degrees C, but frequent after high K(+) depolarization. High pressure freezing of acute brain slices followed by freeze-substitution demonstrated that synaptic spinules are not induced by chemical fixation. These results indicate that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may represent an aspect of membrane retrieval during synaptic activity.

Calcium‐Activated Proteolysis of Neurofilament Proteins in the Squid Giant Neuron

Abstract: The phosphorylation and proteolysis of squid neurofilament proteins by endogenous kinase and calcium‐activated protease activities, respectively, were studied. When axoplasm was incubated in the presence of [γ‐32]ATP, most of the phosphate was incorporated into two neurofilament proteins: a 220‐kilodalton (NF‐220) and a high‐molecular‐weight (HMW) protein. When this phosphorylated axoplasm was subjected to endogenous calcium‐activated proteolysis, two significant phosphorylated fragments were generated, i.e., a soluble 110K fragment and a pelletable 100K fragment. Immunochemical and other analyses suggest that the pelletable 100K fragment contains the common helical neurofilament rod region and that the soluble 100K protein is the putative side arm of the NF‐220. In contrast, neither the HMW or the NF‐220 was detected in the region of the stellate ganglion which contains the cell bodies of the giant axon. However, this region did contain a number of proteins that were sensitive to calcium‐activated proteolysis and reacted with a monoclonal intermediate filament antibody. This intermediate filament antibody reacts with most of the axoplasmic proteins that copurify with neurofilaments, i.e., in the order of their intermediate filament antibody staining intensity, a 60K, 65K, 220K, and 74K protein. In the cell body preparation, the intermediate filament antibody labeled, in order of their staining intensity, a 65K, 60K, 74K, and 180K protein. In both the axoplasmic and cell body preparations, endogenous calcium‐activated proteolysis generated characteristic fragments that could be labeled with the anti‐intermediate filament antibody.

Homer is concentrated at the postsynaptic density and does not redistribute after acute synaptic stimulation.

Homer is a postsynaptic density (PSD) scaffold protein that is involved in synaptic plasticity, calcium signaling and neurological disorders. Here, we use pre-embedding immunogold electron microscopy to illustrate the differential localization of three Homer gene products (Homer 1, 2, and 3) in different regions of the mouse brain. In cross-sectioned PSDs, Homer occupies a layer ∼30-100nm from the postsynaptic membrane lying just beyond the dense material that defines the PSD core (∼30-nm-thick). Homer is evenly distributed within the PSD area along the lateral axis, but not at the peri-PSD locations within 60nm from the edge of the PSD, where type I-metabotropic glutamate receptors (mGluR1 and 5) are concentrated. This distribution of Homer matches that of Shank, another major PSD scaffold protein, but differs from those of other two major binding partners of Homer, type I mGluR and IP3 receptors. Many PSD proteins rapidly redistribute upon acute (2min) stimulation. To determine whether Homer distribution is affected by acute stimulation, we examined its distribution in dissociated hippocampal cultures under different conditions. Both the pattern and density of label for Homer 1, the isoform that is ubiquitous in hippocampus, remained unchanged under high K(+) depolarization (90mM for 2-5min), N-methyl-d-asparic acid (NMDA) treatment (50μM for 2min), and calcium-free conditions (EGTA at 1mM for 2min). In contrast, Shank and calcium/calmodulin-dependent kinase II (CaMKII) accumulate at the PSD upon NMDA treatment, and CaMKII is excluded from the PSD complex under low calcium conditions.