CONFERENCE COVERAGE SERIES
Society for Neuroscience Annual Meeting 2002
Orlando, FL, U.S.A.
02 – 07 November 2002
Synapses Sizzle in Limelight of Symposium Preceding Neuroscience Conference, Orlando: Day I
It would seem intuitively appealing that the brain’s billions of minuscule information exchanges-the synapses-may be the main site of destruction in Alzheimer’s disease. After all, if no more transmitter buzzes across the narrow cleft and no more currents crackle on the receiving side, memories, learning, and other functions must surely disappear. Even so, surprisingly little is understood about what happens to synapses in the years preceding frank Alzheimer’s. This is beginning to change, however. Here in Orlando, a two-day satellite event to the main Neuroscience conference drew 140 researchers to focus attention on just this question. Featuring 22 talks and 45 posters, the symposium brought together basic synapse biologists with neurodegeneration researchers in an attempt to fire up some excitatory activity in the field and trigger new research projects. The journal Neurobiology of Aging sponsored the event, titled "Molecular and Cellular Basis of Synaptic Loss and Dysfunction in Alzheimer’s Disease."
Paul Coleman (University of Rochester), who co-chaired the conference, said that while certain populations of neurons clearly die in Alzheimer’s, they don’t disappear quickly, extinguishing well-functioning synapses overnight. Instead, he said, synapses probably fade away slowly long before the cell dies. The gradual dysfunction and structural disintegration of synapses deserves study in its own right independent of neuronal loss, which has received the bulk of attention to date.
To support his contention, Coleman reviewed studies in his and other labs showing that living neurons of people with AD are already losing synapses. Older studies showed that the dendrites of neurons in AD shrink first at the tips and then further towards the cell body while the neuron is still alive. Others showed that those regressing dendrites contain fewer synapses than do dendrites of normally aging brains. More recent work in Coleman’s lab showed that neurons that are filled with tangles in people with AD express much smaller amounts of certain synaptic markers than do neighboring neurons that have no tangles yet. Taking the same experiment a step earlier, Coleman said that even neurons from AD patients that have no tangles yet express lower levels of synaptic marker proteins than comparable neurons from normally aging people, suggesting that synapses decline in AD before classic pathology becomes visible.
Other work amplifying mRNA from individual neurons and analyzing it on microarrays identified a slew of synaptic proteins whose expression drops in still-living AD neurons, including dynamin, the adapter protein AP3MuB, and others. Interestingly, Coleman’s group found that genes involved in synaptic function decline even while structural synaptic markers remain normal, suggesting subtle synaptic dysfunction as an early sign of Alzheimer’s.
Most of the important questions remain wide open for study, said Coleman. This includes the mystery of what starts this decline, what its sequential steps are, and how these steps relate to pathology and cognitive function. Also unclear is why cholinergic neurons are supremely vulnerable while GABAergic neurons, for example, are relatively spared. Finally, it is also unclear whether synaptic loss precedes neuronal loss and symptoms in other neurodegenerative diseases.
Even so, one thing seems clear. "Cognitive decline is largely a function of the decline of synapses. The therapeutic notion that follows is, we should try to rescue the synapses, not just neurons. Once the synapses are lost, what’s the point in saving the cell body?" Coleman said.
What, then, should the intrigued AD investigator study? The remaining talks of the first day described different aspects of basic synapse biology that introduced some of the "whos" and "hows" of synaptic plasticity. Together, they offered numerous angles and candidate molecules to scrutinize in neurodegeneration studies. For example, co-chair David Bredt of the University of California, San Francisco, described the postsynaptic density (PSD) of the typical glutamatergic synapse. So named years ago because it appeared in electron microscopy as a stripe of fuzzy black dirt just below the nice clean line of the postsynaptic membrane, his dynamic organelle contains roughly 100 proteins. Especially prominent is a large, 30-protein signaling complex of glutamatergic AMPA and NMDA receptors, sodium and calcium channels, and anchoring and adaptor proteins for these channels called GRIP, homer, PSD-95, shank, as well as signaling proteins such as CaM kinase 2, immediate early genes such as arc, and c-fos, cell adhesion and cytoskeletal proteins (Kennedy, 2000).
Many of these proteins are highly dynamic; they respond to synaptic activity by turning on genes, recycling and trafficking receptors. For example, Bredt described studies of a protein underlying a spontaneous mutant mouse strain kept at Jackson Labs in Bar Harbor, Maine, that was dubbed stargazer because it stares upwards during epileptic seizures. Stargazin, it turns out, recruits intracellular AMPA receptors to the membrane and chaperones their glycosylation in the ER. All this happens in the dendrite, into which the ER extends. PSD-95 then recruits extrasynaptic receptors to the synapse, and both PSD-95 and stargazin activity changes with synaptic stimulation in ways that remain poorly understood, Bredt said. The stargazin variety in the unfortunate mouse strain is active in cerebellar granule cells, but one family member clusters AMPA receptors in glial cells, another in hippocampus, Bredt said.
Oswald Steward of UC Irvine outlined work into the poorly understood question of how the synapse communicates with the nucleus as a synaptic activity is being consolidated into a lasting memory. "There must be signaling from the synapse to the nucleus to turn on gene expression. Newly synthesized gene products must then be delivered back to the active synapses and changes take place over time that create an enduring alteration in synaptic efficacy," Steward said.
To get at this problem, Steward first pointed out that the base of dendritic spines (synapses ring the top of these spines) contains membranous polyribosomes that translate mRNAs. James Eberwine of the University of Pennsylvania reported that up to 600 different mRNAs get transported from the nucleus to these spines, and Steward finds about 20 of those in high abundance. These include cytoskeletal proteins, kinases, MaP2, calmodulin, G-protein, BDNF, and the many other components of the PSD signaling complex.
Consider the immediate early gene arc, for example. Synaptic activity induces its mRNA expression; in the hippocampus, a single large stimulus involving NMDA receptor activation strongly turns on its expression, said Steward. Interestingly, arc mRNA then is transported only to those synapses that were active, and accumulates there within two hours, about the time period during which consolidation takes place. In addition, Steward said, arc mRNA that is already present in the dendrite at the time of stimulation gets targeted to active synapses within minutes.
How does the call for arc expression reach the nucleus? Steward’s research favors the MAP kinase pathway in such a way that NMDA activation would trigger Erk phosphorylation (Erk is a downstream member of this pathway and translocates to the nucleus when activated.) The targeting of the resulting mRNA back to active synapses, however, works through a different signal, Steward said, perhaps CAM kinase 2, and further stimulation of the requisite NMDA receptors then trigger docking of arc protein to the postsynaptic density. He suggested this sequence as a possible pathway for how repeated stimulation can dynamically change a given set of synapses, but he added that it remains unknown whether and how these signaling mechanisms change in age-related memory disorders.
Eberwine expanded on Steward’s reference to mRNA transport and translation in the dendrites. Dendrites make up 90 percent of the cell surface of many neurons, he said, so it is not surprising that incipient neurodegeneration would begin there. Eberwine reviewed the clever methods his lab developed to remove individual dendrites from cultured primary hippocampal neurons, amplify their mRNA content, and screen microarrays to identify them.
To show that these mRNAs are actually translated into protein locally in the dendrite, his group sprayed tagged mRNA directly into dendrites and then detected its protein with antibodies. They developed a GFP marker for local translation in the dendrites, as well. Moreover, they found that regions in the mRNAs and in the local ribosomes control the particular characteristics of dendritic translation, which differ from those in the soma. Which mRNAs are translated in dendrites? One example is glutamate receptor mRNA, which is locally made and then inserted into dendritic membranes.
How do the mRNAs get into the dendrites? Signal transduction is not enough, since nucleic acids do not float unaccompanied through the cytoplasm. Eberwine showed the mRNAs travel with RNA-binding proteins, such as translin or PABP. However, it remains unknown which RNA-binding proteins bind which cargo RNAs, or how this binding controls transport. While this area is in its infancy, Eberwine stressed that RNA-binding proteins’ ability to modulate the transport and distribution of particular mRNAs will make RNA-binding proteins important in understanding neurodegenerative diseases. One example exists in their role in Fragile-X, the most common form of mental retardation, and Eberwine recommended that researchers look for differences in the functioning of these proteins in Alzheimer’s.
Paul Worley of Johns Hopkins School of Medicine in Baltimore, Maryland, reviewed the function of three immediate early genes that function at the excitatory synapse. Narp (neuronal activity-regulated protein) is made and secreted into the excitatory hippocampal synapse, peaking at about 16 hours after strong stimulation (typically, immediate early proteins peak at two hours). Narp expressed in one cell can cluster AMPA receptors on a contacted cell. In this mechanism of synaptic plasticity, narp can act as a presynaptic aggregator for postsynaptic receptors. In this sense, narp is a cousin of agrin, a molecule well-studied for its key role in the clustering acetylcholine receptors on the developing neuromuscular junction. Similarly, NP1 is another immediate early gene that induces AMPA receptor clusters. NP1 and narp can dimerize and form mixed hexamers that can cluster AMPA receptors more stably.
A member of a rapidly growing family, the immediate early homer plays a role in the calcium dynamics and G-protein signaling of the metabotropic glutamate receptor (mGluR). Together with arc, homer is the most dynamic of all known immediate early genes in neurons, Worley said. Homer is both a scaffolding protein and a catalyst of receptor function. It has an EVH1 domain, much like mena, Wasp, and other proteins known to be able to change the assembly of the actin cytoskeleton that runs just underneath the dendritic membrane. Homer also is able to crosslink the mGluR receptor with the endoplasmic reticulum, meaning that this organelle comes into very close contact with the postsynaptic density, extending right to the top of the dendritic spines.
Interestingly for Alzheimer’s, perhaps, introducing homer into dendrites of cultured neurons produced a receptor clustering that dramatically increased calcium influx in response to stimulation, Worley said. Intracellular calcium signaling and homeostasis are known to play a role in APP processing (see further below).
Regarding the immediate early gene arc, Worley said that it truly is a postsynaptic density protein that may be a chaperone or scaffold for other proteins bearing an SH3 domain. In addition to the ERK-mediated transcription Steward described, Worley said his lab detects arc transcription as early as three minutes after a mouse is beginning to explore a new cage.
While in general, immediate early genes are thought to enhance synaptic transmission, there are also indications that they can dampen transmission following a large stimulus. How these roles relate to neurodegeneration is still unclear, Worley added.
Howard Federoff of the University of Rochester, New York, presented the newest results of his ongoing studies of NGF. Federoff said NGF is much more than a developmental survival factor for neurons. He believes it is a key regulator of synaptic plasticity, in part because it is present on the postsynaptic side, its expression is regulated by activity, and its release from hippocampal neurons and dendrites is controlled by depolarization.
Federoff’s lab created inducible NGF transgenics. The mice develop normally while the transgene is dormant; then the researchers can express it in any brain region by injecting a Herpes simplex-derived activator. Federoff’s group turned the NGF on in the hippocampus of young adult mice to study its effect on cholinergic input into the hippocampus from the basal forebrain. This septohippocampal projection in the mouse corresponds to the human perforant pathway going from layer two of the entorhinal cortex to the molecular layer of the dentate gyrus in the hippocampus, which is involved in learning and degenerates early in Alzheimer’s. It turned out that mice with gain of NGF function in their hippocampus are better at special learning than are controls. (Incidentally, spatial learning is the behavioral category that generally shows the most robust results, i.e., decline, in APP-transgenic mice, as well.) The mice had extensive spatial reorganization of the cholinergic fibers into the hippocampus, and the extra NGF further strengthened this effect.
Federoff also reported that his group ran microarray tests of gene activation in the NGF-enhanced hippocampus compared to the control side, and of the septum area that projects to the hippocampus. (The NGF released by hippocampal neurons is taken up and transported back to the septal neurons.) He is currently analyzing genes whose expression changed; these include signaling molecules, synaptic proteins, ion channels, and transcription factors.
Finally, Federoff described some unpublished work on NGF expression in cultured primary neurons. Almost all of the NGF they saw was in the postsynaptic density. Apparently, the neuron maintains two pools of NGF. A constitutive pool in the soma may be the one that maintains survival, and a regulated pool in the dendrites may be the one that responds to activity by the presynapse. When NGF levels rise in the neuron, it re-apportions the relative amounts in those pools.
Mark Bear of Brown University, Rhode Island, presented work on long-term depression (LTD), a negative form of synaptic plasticity usually induced by stimulation of NMDA receptors. In LTD, the expression of AMPA receptors and their number in the synaptic membrane declines. Protein kinase A, the phosphatase 2B, and the anchoring protein AKAP 150 mediate this loss and could be checked for changes in Alzheimer’s, Bear said.
In a demonstration of what "Use It or Lose It" means in molecular terms, Bear also reported on work with monocular deprivation. Traditionally, shutting off sensory input from one eye has been used to study synaptic pruning and activity-based refining of connections in the visual cortex during a particular sensitive period in postnatal development. Temporarily covering one eye is also used to help correct certain eye asymmetries in children. Here, Bear asks if the known molecular mechanisms of LTD underlie changes at postnatal synapses that have fallen silent. When Bear analyzed synaptic currents in the visual cortex from rats kept with a closed eye, he found a signature typical of LTD. Not only were there 20 percent fewer synaptic AMPA receptors in slices cultured from rats after only 24 hours of deprivation, but also, the GluR1 subunit of AMPA receptors had a telltale dephosphorylation at serine 845. Previously, Bear had reported dephosphorylation of this site by cAMP-dependent PKA in in-vitro experiments with pharmacologically induced LTD.
What this might mean for Alzheimer’s is unclear. Bear said that while in postnatal life, LTD is the effect of insufficient activity, in aging it might occur in response to inappropriate activity. Coleman replied that the rapid time course seen in Bear’s study does not jibe with the presumably gradual decline of synapses in the degenerating brain; however, the mechanisms of LTD deserve exploration because it is especially prominent during two periods of life, namely postnatal development and aging.
Edward Ziff of New York University reported that palmitoylation of AMPA receptor-binding proteins is a newly appreciated mechanism by which these proteins control the trafficking of AMPA receptors between the synapse and other intracellular vesicle-like structures. He introduced PICK as an AMPA receptor-binding protein that may be involved in LTD by dissociating the Glur2 AMPA receptor from the membrane; a protein called NSF antagonizes this action. Ziff described other work with protein complexes called SNARE and SNAP, which determine the mobility of AMPA receptors.
Switching gears, Mike Ehlers of Duke University in Ithaca, New York, described recent research into how groups of proteins of the postsynaptic density change in response to synaptic activity. The talk visibly fascinated the audience with its implication of the proteasome as a force behind these changes, and not the lysosomal system, which has previously been implicated in synaptic protein turnover.
Ehlers said that while most of the components of the PSD are known, their relative abundance was not, nor was there a global understanding of the dynamic changes within them. He said the PSD is so highly plastic that many components reorganize within minutes of activity; lasting changes in activity alter the strength of the synapse, the number of receptors in it, as well as the size-and even appearance and disappearance-of the PSD.
To assess multiprotein changes, Ehlers developed a method to isolate, from cultured neurons, PSDs that were in a similar state of activity, and analyzed their protein content with gel density measurements. Following increased synaptic activity, he saw discrete groups of proteins go up and down in concentration, and the mirror image of that occurred following blocking. These changes reached a plateau after one day and reversed when the activating or blocking drugs were washed out. Overall protein content did not change.
To understand further some of the underlying mechanisms-in addition to the translational and transcriptional findings as described by Steward and Eberwine-Ehlers looked at protein turnover with pulse-chase experiments. He found that the proteins of the PSD are normally turned over every five hours; again, synaptic activity sped this up and blockade slowed it down.
What might be controlling this protein turnover? One candidate is the ubiquitin-proteasome system. It is not clear how it affects synapses, but it recently has been implicated in LTP and synapse development, Ehlers said. With fractionation experiments, he found that he could detect ubiquitin conjugation (the step marking proteins for degradation) in fractions containing synaptic proteins, and this ubiquitin labeling increased with synaptic activity.
These data, while preliminary, may open a new aspect for the role of the proteasome in neurodegenerative diseases. What if the proteasome slows down too much? Could that impair the ability of the postsynaptic side to meet the demands posed by signals coming from the presynaptic side?
If indeed proteasome activity declines in the run-up to AD, one protein that might accumulate is tau. Eva-Maria Mandelkow of the Max-Planck Institute in Hamburg, Germany, updated her and her husband’s hypothesis that tau’s role in AD pathogenesis might derive from its blockade of axonal transport, starving the synapse of vital ingredients including APP (see previous news story). Even very minor elevations of tau concentration, over time, might cause neurodegeneration in this way, Mandelkow said.
Jesus Avila of University of Madrid, Spain, described a conditional transgenic mouse in which overexpression of GSK3, one of numerous kinases known to phosphorylate tau, can be induced with tetracycline. In these mice, tau was hyperphosphorylated, but tangles did not form. Avila suggested that the cytoskeleton would change as hyperphosphorylated tau is no longer able to stabilize microtubuli. This concludes the first day of presentations delivered at the Neuroscience Conference in Orlando. Please see Day 2 summary.-Gabrielle Strobel.
References
News Citations
- Tau Accused of Blocking Transport, Causing APP to Linger and Nerve Processes to Wither
- Synapses Sizzle in Limelight of Symposium Preceding Neuroscience Conference, Orlando: Day 2
External Citations
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Synapses Sizzle in Limelight of Symposium Preceding Neuroscience Conference, Orlando: Day 2
William Honer of the University of British Columbia, Vancouver, reviewed the existing literature on synaptic pathology in Alzheimer’s as viewed from the perspective of presynaptic markers. As usual in this field, the findings are contradictory. While 10 years ago, the relationship between the levels of the marker synaptophysin and cognitive impairment seemed clear, today the picture is more mixed, Honer said. He asked the following questions:
- Are all presynaptic proteins equally affected in Alzheimer’s? Six studies comparing the same markers (mostly synaptophysin, syntaxin, SNAP-25, synaptotagmin, VAMP) in different brain areas suggest that, no, different proteins go down in concentration at different times. He cautioned, however, that as a rule, existing research on these proteins remains far less sophisticated than that done on tau, for example.
- Are all brain regions equally affected? As expected, the hippocampus, temporal and frontal cortex are most affected. Eighteen studies have confirmed this.
- What is the relationship with pathological markers? In the perforant pathway, the correlation of synaptophysin with plaque number is weak, with tangle number a little stronger. Of 21 studies on the subject using other presynaptic markers, most find the same result. Some studies relate a decline in presynaptic proteins to soluble Aβ, glutamate transporter loss, and microglial activation.
- What is the relationship with cognitive impairment? Most of the 16 existing studies on this question found a link between cognitive testing and loss of presynaptic proteins. However, the simplicity here deceives. In mild dementia, presynaptic proteins tend to be increased relative to control, reflecting, perhaps, a temporary compensatory effort by the brain. In other mild cases, Mini Mental State Exam scores did not correlate with frontal cortex synaptophysin.
- What is the relationship with phase of illness? A large study (Tiraboschi et al., 2000) looking at synaptophysin showed a small, insignificant difference in the mildest cases. Choline acetyl transferase levels did not differ statistically in mild cases, either. (See also related news.) Honer said that his group, as well, did not find a robust decrease in synaptophysin, syntaxin, or SNAP-25 levels until the more severe stages. In all, 11 studies on the question did not find consistent results.
Honer cautioned that, while this last finding might seem to weaken the notion that synapses die very early on in AD, it might also reflect inconsistency in the ways mild cases are assessed and diagnosed. As an interesting aside, Honer mentioned ongoing work showing that antibodies against presynaptic proteins can visualize plaques almost as well as standard methods, such as thioflavin S. VAMP, especially, is highly present in plaques. Finally, Honer threw out another bit of food for thought: In APP23 transgenic mice, the concentration of most presynaptic proteins assessed showed a temporary increase at 12 months, followed by a decrease below, but not far below, control levels. All of this fits the speculative notion that the pathology comes first, and sometime later, once synaptic damage begins, cognition suffers. Most people do not get diagnosed until this has progressed quite far.
Valina Dawson of Johns Hopkins University, Baltimore, Maryland, laid out a series of studies aimed at answering the question of how specific populations of neurons die. She suspected death pathways other than the caspase pathways were at work in neurodegeneration, in part because classic apoptotic players were first described in cell types that are programmed to turn over frequently. Neurons, by contrast, are designed to stay alive through a person’s lifespan and get replaced very slowly, if at all.
Dawson described elucidation of the following death pathway: Glutamate overly excites NMDR receptors; this activates neuronal nitric oxide synthase. Nitric oxide diffuses and, together with superoxide anion produced in mitochondria, leads to the formation of peroxinitrite radicals, damaging DNA and thus triggering expression of the nuclear protein PARP. This enzyme somehow causes the release of apoptosis-inducing factor (AIF) from the outside of mitochondria, where it is normally anchored. AIF then translocates to the nucleus and induces nuclear condensation. AIF, then, is the endpoint of a caspase-independent mode of cell death. This new pathway is an alternative to death by caspase-cytochrome C, Dawson pointed out.
She further described work using the MPTP model of Parkinson’s to suggest that this synapse-mediated mechanism of cell death might indeed be active in disease, and suggested a focus on developing therapeutic agents that bind AIF and redirect it away from the nucleus and toward proteasomal degradation. (See also related news; related news.)
Roberto Malinow, at Cold Spring Harbor Laboratory in Long Island, New York, presented new work probing a potential normal function of Aβ. Could it be that the peptide, once released from the synapse, acts as a negative feedback signal to keep neuronal hyperactivity in check? This might normally occur under conditions of high activity, but become dysregulated when APP or Aβ concentrations rise, Malinow said. His student Flavio Kamenetz prepared organotypic slices of transgenic mice carrying the APP Swedish mutation, cultured it with high or low activity, and then analyzed the Aβ content with an ELISA. He found that a decrease of activity with tetrodotoxin or a benzodiazepine led to a decrease of Aβ content by half; an increase of activity with the substance PTX correspondingly increased Aβ in medium.
By analyzing the different APP cleavage products, Kamenetz found that the BACE reaction is the controlling step. Then the scientists expressed GFP-APP delivered to neurons in the slice by injection of a viral construct and recorded the electrophysiological output of synapses from single cells to ask how extra APP affected their activity. Seen in the dendritic spines, the APP depressed excitatory transmission of AMPA and NMDA receptors, though the GABA transmission remained unaffected, Malinow said. Expressing a form of APP with a mutation in the Aβ sequence that prevents formation of the peptide abrogated the depression, as did γ-secretase inhibitors. This indicates that Aβ, not APP, mediates the synaptic depression. Other APP cleavage products were not necessary to produce the effect.
The interaction is a bit circular: Aβ leads to synaptic depression, and synaptic activity leads to higher Aβ secretion, Malinow reported, probably via an effect on BACE. Kamenetz also recorded from individual neurons surrounded by infected, Aβ-releasing cells and compared its transmission with that from a neuron surrounded by noninfected cells. Lo and behold! Transmission was down in the noninfected region, indicating that secreted Aβ acts on neighboring cells.
In hippocampal slices of normal, non-transgenic mice, this phenomenon is visible only after intense stimulation. Under conditions of strong LTP activation, Aβ depresses transmission somewhat and presenilin-inhibitors increase transmission, Malinow said.
How could this play out in AD? Malinow said that two speculative scenarios came to mind. An unknown factor causes an increase in the amount of Aβ, and the synapses disappear once they have been repressed long enough. Again, use it or lose it. Alternatively, synapses could burn out and crash. Conditions of prolonged intense excitation would induce intense APP processing, and the Aβ effect might somehow turn into a positive feedback signal. David Holtzman commented that this latter notion would fit well with studies finding that synaptic markers go early in transgenic mice and also are not decreased overall in early cognitive impairment. It also fits with clinical data that people who die with pathology but without dementia symptoms do not yet have synaptic changes.
Lennart Mucke, University of California, San Francisco, focused on transgenic mice studies suggesting how Aβ accumulation (but not plaques), aging, and ApoE isoform might lead to cognitive dysfunction. His lab has a paper coming out in the November 15 J. Neuroscience on the topic, which Alzforum will summarize in this space, and other research on calcium regulation and behavioral changes in transgenic mice will appear later.
David Holtzman of Washington University in St. Louis, Missouri, asked how Aβ could possibly cause LOAD when there is no obvious overproduction of the peptide in this vast majority of cases. PDAPP mice allow him to address this question, since they also do not have a longstanding gradual buildup of Aβ early in life. Aβ increases only when plaques start forming. "Why does Aβ convert to forms that are toxic?" Holtzman asked. He then reviewed work on factors influencing Aβ fibrillization that was recently summarized on Alzforum (see news story; news story).
In addition, Holtzman presented new ways to study the dystrophic, swollen neurites that occur around amyloid plaques of APP-transgenic mice. Holtzman crossed with PDAPP mice a strain of YFP-transgenic mice that express yellow fluorescent protein throughout the neuronal cytosol, reaching into distal neurite tips. He found that the mice have numerous swollen neurites, but only where there are mature plaques. Holtzman emphasized how widespread the occurrence of swollen neurites in an areas containing amyloid, suggesting that neuritic dystrophy, i.e., synaptic damage in still-living neurons, is much more extensive than previously thought. Preliminary studies of organotypic slice cultures of these mice indicate that this method can visualize dendrites, dendritic spines, and axons, and observe them over a period of several days.
Frank LaFerla of the University of California, Irvine, gave a whirlwind tour of the molecular, pathological, and electrophysiological characterization of his new triple-transgenic mouse model of AD. LaFerla tried to recapitulate more of the AD pathology than do current models, and do so with a method that avoids the confounding effects that can occur after breeding strains from different backgrounds. Instead of crossing strains, LaFerla took a mutant human PS1 knockin mouse developed by Mark Mattson and injected into its single-cell embryos both a mutant human tau and a mutant human APP transgene. Luckily, both transgenes inserted into the same site, enabling the mice to breed as if they were a single strain, said LaFerla.
Tau and APP are expressed in the CNS, mostly in AD-vulnerable regions, and in the spinal cord, and Aβ accumulates with age, LaFerla said. Aβ accumulation inside neurons at six months of age is the first detectable sign of pathology, LaFerla added, saying this supports a role of cytoplasmic Aβ early in the disease. By nine months, extracellular deposits show up in cortex and hippocampus. At six months, tau is not visible with immunoreactivity (not just tangles; there is no tau pathology at all), but by 12 months, initial signs of tau pathology appear, and by 15 months, the mice have extensive tau. "We believe that Aβ accumulation is the initiating trigger for sporadic and familial AD. We think Aβ is upstream of tau in the pathologic cascade," LaFerla said.
Electrophysiologic recording from the CA1 region of the hippocampus showed deficits in synaptic transmission and in LTP even at six months, when there was no extensive pathology yet. At one month of age, these parameters were normal. This leads LaFerla to propose that synaptic deficits occur very early in this mouse.
What could be the molecular mechanisms underlying this? LaFerla described experiments to tease apart the effect of presenilin mutations on Aβ generation from their other effect on disrupting calcium homeostasis (see related news story). LaFerla described some unpublished data suggesting that the intracellular tail of APP, AICD, affects the transcription of the calcium-related gene SERCA, which further increases calcium levels in ER stores already overfilled as a consequence of presenilin. This might further increase Aβ levels. LaFerla ascribes the age-dependence of LOAD to the waning activity of Aβ-degrading enzymes. (LaFerla; related news).
Virginia Lee of the University of Pennsylvania, Philadelphia, urged the audience to consider a link between tau and α-synuclein (see related news). She also reported that an α-synuclein-transgenic mouse recently developed in her lab (see related news) unexpectedly showed tau pathology as well, suggesting that the two proteins might interact and facilitate each other’s pathology.
Robert Edwards of University of California, San Francisco, reported that a small proportion of α-synuclein occurs in lipid rafts, specialized membrane regions that are high in cholesterol and are also known to harbor APP and Aβ. One of the two α-synuclein mutations known to cause familial, early onset Parkinson’s disease, A30P, abrogates this localization.
In the final talk, Don Price of Johns Hopkins University pulled together recent findings in his division on BACE around the theme of how Aβ secretion from synapses could damage synapses. He suggested a model where Aβ deposits around synapses, the presynaptic side detaches and swollen neurites form, glia proliferate and move in, and the axon degenerates. One set of findings supporting this notion, Price said, was that APP, its cleaving enzymes, and Aβ all are found in and around terminals and neurites. Price, with Vassilis Koliatsos, and Sam Sisodia’s group each have a paper coming out on this topic in J. Neuroscience on November 15, which Alzforum will cover in this space.
Price summarized knockout studies of the components of APP processing. Mice lacking nicastrin or presenilin 1 die in utero or shortly after birth, respectively, but BACE knockouts are healthy and breed well. Even when crossed with APP/PS1 transgenic mice, BACE knockout mice produce no Aβ at all, Price said. Preliminary evidence shows that these mice perform as well in the Morris water maze as do wild-type mice, even though they massively overproduce APP and APP/PS-1 mice that do have BACE perform poorly, Price said. This adds to existing evidence making a case for the development of BACE inhibitors, which are being pursued in most major pharmaceutical companies, said Price. He did not reveal any information on their status.
Price said that the expression levels of the APP-processing enzymes reveal why Alzheimer’s is a brain disease even though all cells make APP. α-secretase, which prevents Aβ generation by cutting in the middle of its sequence, is barely expressed in neurons but abundant in peripheral organs, as is BACE 2, which also makes an amyloid-preventing cut. BACE1, however, is abundant in brain but few peripheral organs checked so far. (The pancreas expressed BACE 1 but there, alternative splicing creates an isoform that cannot create Aβ.) See news story; news story; news story; and news story.
The symposium ended with the drawing of a bottle of champagne as the prize for filling in an evaluation. Allen Butterfield of the University of Kentucky, Lexington, was the lucky winner. The only pet peeve about the symposium may have been that after two days of unrelenting excitation of the audience’s collective synapses, everyone could have used a rejuvenating glass of fizz. All talks in this event were interesting. Those that did not get much space in this on-site summary were either covered on Alzforum previously, or were presented when the writer’s brain, regrettably, had temporarily filled up! Since there was no time for fact-checking, I especially invite all speakers to send corrections to gabrielle@alzforum.org.—Gabrielle Strobel
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References
News Citations
- Revise Mechanism for Alzheimer's Drug? Acetylcholine Increased in Earliest Stage of Disease
- Oxidative Stress Triggers Neuronal Cell-Cycle Reentry
- In C. Elegans, Aspartyl Proteases and Calpain, Not Caspases, Play the Grim Reaper
- Ocean Waves Stimulate Brain Waves: Meeting Report from Caribbean
- Stockholm: Clusterin (or Beware the Evil Chaperone!)
- Society for Neuroscience Annual Meeting: The Calcium-Presenilin Connection
- Does the Intracellular AβPP Fragment Regulate Calcium?
- Meeting Report: American Society for Neurochemistry (22-26 June 2002)
- Presenting: A Not-Quite-Parkinson's Mouse Model
- BACE Above Base in Alzheimer’s Patients
- More on BACE1, the New Darling of Alzheimer's Disease Drug Developers
- Another Not-Quite-Parkinson's Model
- β-Secretase a Clean Therapeutic Target
Other Citations
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Orlando Conference: Hyperphosphorylation—It's Not Just Tau's Problem Anymore; APP, Too?
Regulated intramembranous proteolysis, RIP for short, denotes an unusual way of cutting proteins lodged in a membrane right inside their membrane-spanning parts. This process of proteolysis, smack in the middle of a lipid bilayer, was considered extremely rare, with only one prior example in the cholesterol biosynthesis pathway, until recently, when the workings of γ-secretase became more clear. Now, RIP is receiving a large amount of attention in the Alzheimer’s field, as researchers bear down on the details of how this complex process occurs and is controlled by multiple proteins. The hope is that a deeper understanding of the regulatory events and players involved in APP proteolysis will yield additional drug targets to pursue should current efforts to develop direct γ-secretase inhibitors fail.
In a symposium on the topic, Li-Huei Tsai of Harvard Medical School presented a new aspect of APP proteolysis regulation. She said that one way in which APP proteolysis might be controlled inside the cell lies in phosphorylation of APP. She detailed a series of studies that found eight phosphorylation sites on the intracellular part of APP, which, once cleaved in the reaction that also frees Aβ, translocates to the nucleus and is thought to affect gene expression there.
Asking which phosphorylation event could influence Aβ production, Tsai’s team identified threonine 668, and found, with Cindy Lemere at Brigham and Women’s hospital, that it is selectively phosphorylated in the hippocampal neuropil of postmortem sections of human AD brain and APP-transgenic mice, but not various controls. Two structures carrying this phosphorylation stood out: dystrophic neurites and granular vesicles.
Since the same area also had robust neurofibrillary pathology, Tsai et al. looked for possible interactions and found that APP phosphorylated on threonine 668 occurs in those neurons that also carry hyperphosphorylated tau. Biochemical and mass spectrometry experiments indicated that as many as seven of the eight phosphorylation sites of the cytoplasmic APP tail may be phosphorylated in AD samples; some were hyperphosphorylated.
In cultured hippocampal neurons, phosphorylated APP did not co-localize strongly with overall APP, but phosphorylated APP and BACE1 both appeared to reside in the growth cones, and they were on endosomal vesicles, not in the lysosomal pathway, Tsai reported. With Rachael Neve at McLean Hospital in Belmont, Massachusetts, Tsai’s group conducted further experiments with rat primary neurons infected with various viral APP constructs to ask whether threonine 668 phosphorylation affects Aβ production, and indeed, preliminary experiments suggest it may do so.
Taken together, the data led Tsai to suggest a working hypothesis. As APP matures and moves through the secretory pathway of consecutive membrane compartments, hyperphosphorylation of its cytoplasmic domain might influence the decision point where APP will either undergo cleavage by α-secretase and become inserted into the cell membrane, or will be cleaved by BACE1 in endosomal vesicles, leading to Aβ generation. Tsai invited the audience to consider whether, perhaps, phosphorylation on threonine 668 somehow directs APP toward the endosomal compartment, favoring cleavage by BACE.-Gabrielle Strobel.
Addendum, added 12 November 2002.
A look in the literature revealed that the above news story, written on-site right after the presentation, omitted mention of previous literature about APP phosphorylation. In fact, Tsai had pointed out that New York researchers including Paul Greengard, Joseph Buxbaum, Sam Gandy, and others have studied this question for the past 12 years, and other labs are pursuing it, as well.—Gabrielle Strobel
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Orlando: Adding to ADDLs: Where Are They; What Might They Be Doing?
As the amyloid pathology is shifting away from blaming predominantly amyloid plaques to now pointing a second accusing finger to smaller, non-deposited aggregates of the Aβ peptide, several labs are racing to show exactly what these small suspects might be doing, especially around synapses, and just how they might be toxic. For example, Bill Klein of Northwestern University in Chicago has a series of presentations here in Orlando on his lab’s attempt to take the existing data into the context of neurons and in vivo.
His group is extending their 1998 findings that small Aβ42 oligomers, which they call Aβ-derived diffusible ligands, or ADDLs, (see Lambert et al.) are neurotoxic. On Sunday here at the Neuroscience Conference, Kirsten Viola presented a poster suggesting that, on cultured rat embryonic neurons and human neural precursor cells, ADDLs bind specifically and form distinct little clusters. The authors call these dots "puncta," as clusters of synaptic proteins are frequently referred to. (The ADDL puncta did not co-localize with classic synaptic markers such as synaptophysin or the NGF receptor, however.)
At about a quarter of a micron in size, these dots are near the resolution limit of the light microscope, making further structural analysis in that way difficult. However, the clustering seems to fit with findings from Hilal Lashuel and Peter Lansbury that annealed, higher-order structures of Aβ oligomers can form ring-like structures in the electron microscope and their hypothesis that these might insert into neuronal membranes and form pathologic pores (see Lansbury interview).
Viola et al. saw the puncta on all cell surfaces but they appeared particularly dense along the neuron’s processes, including the neurite tips and growth cones. Further, immunocytochemistry, western blots, and immunoblots of membrane preparation from the cultured cells found that the ADDL puncta co-localized with phosphorylated FAK-YP, a protein previously linked to LTP and Aβ toxicity. The presence of ADDLs appeared to increase the number of FAK-YP puncta by about 100 sites per neurons. Finally, the poster reported that ADDLs appear to bind a novel toxin receptor, which remains unidentified.
Klein’s group will present three more posters on ADDLs on Wednesday, so watch this space for continued coverage by our guest writer, Brenda Patoine.
On the question of how ADDLs might pass their neurotoxic signal on inside the neuron, Lennart Mucke’s group also presented a poster yesterday that implicated more deeply the tyrosine kinase Fyn and its downstream pathways. The link had been made in brain slice experiments previously. Crossing human mutant APP-transgenic mice with Fyn knockout mice decreased the synaptic pathology that the scientists see in those Aβ-overproducing mice when Fyn is present.—Gabrielle Strobel
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- Scheinfeld MH, Roncarati R, Vito P, Lopez PA, Abdallah M, D'Adamio L. Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP). J Biol Chem. 2002 Feb 1;277(5):3767-75. Epub 2001 Nov 27 PubMed.
Orlando: New Rat Model Suggests Aβ Alone, Not APP, Is Bad for Rat Smarts
Today at the Society for Neuroscience Conference, Ross Bland’s poster about a new rat model for the study of Alzheimer’s disease drew quite a crowd. Performed in the lab of Matthew During at Thomas Jefferson University in Philadelphia and Neurologix Inc. in Newark, Delaware, the study applied to Alzheimer’s disease the lab’s expertise with adeno-associated virus (AAV), which it had previously directed toward experimental gene therapy approaches. Unlike with genetic transgenic models, in AAV models one injects a viral construct into the desired brain region of an adult animal (at least in the case of AD) and can achieve stable expression of the transgene for years. Bland said one could inject several transgenes within a given DNA size limitation—for example, PS1 and tau, or α-synuclein and tau—and study their interaction without having to worry about the potentially confounding effects of crossing different genetic backgrounds. Unwanted differences in expression in the relevant brain areas are also not a problem with this approach, which obviates the need for costly breeding of transgenic strains, Bland added.
The scientists microinjected into rat hippocampus these constructs: the full-length mutant human APPswe gene, various control constructs and, most importantly, an Aβ gene construct originally developed by Todd Golde called BRI-Aβ42 fusion. It is modified in such a way that Aβ42 gets expressed in the secretory pathway without having to be cleaved first from APP. This allowed a comparison of the effects of APP overexpression, which includes the generation of all its attendant cleavage products, with just Aβ42 alone.
A month later, the rats expressed high (but not yet quantified) levels of Aβ42 in the subiculum, CA1, CA3 and granule cell layers of the dentate gyrus of the hippocampus. The peptide also appeared in the entorhinal cortex, probably because some projecting neurons picked it up in the hippocampus and transported it back. The pathology of these rats is not yet fully analyzed.
The key finding of the study was a robust decline in the performance of Aβ42-expressing rats in the Morris water maze, where they showed deficits at both three and six months after the injection. Rats expressing the full mutant APPswe behaved like the controls in this test. Intriguingly, the poster noted that a trend toward poorer performance showed up three days after injection, but that data was not shown. Moreover, the Aβ42-expressing rats also showed a clear deficit in a water maze test of working memory.
Curiously, both the APPswe- and Aβ42-expressing rats were much more physically active than were controls, but it is not clear whether they ran around more because of anxiety or because there really is some mysterious effect of these transgenes on locomotor activity, Bland said.—Gabrielle Strobel
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Orlando Conference: The Chicken <I>and</I> the Egg
Aβ levels and amyloid plaque load can be reduced significantly in animal models of Alzheimer's disease by interfering—either genetically or pharmacologically—with the immunoregulatory molecule CD40 ligand, according to a report in the current Nature Neuroscience by Jun Tan, Terrance Town, Michael Mullan, and colleagues at the University of South Florida in Tampa and at Yale University in New Haven, Connecticut. Follow-up findings are also being presented here at the Neuroscience Conference-see below.
Previous research led by Mullan and others (see related news item A; and related news item B) had indicated that Aβ upregulates levels of the CD40 receptor on the surface of microglia, and that CD40 interaction with its ligand (CD40L) played a role in the activation of microglia. As might be expected, however, the cause-and-effect relationship is not so simple, because CD40 and CD40L are then found in and around amyloid plaques in AD brain, suggesting that they may play a subsequent role in Aβ production and amyloidogenesis. The question of whether microglial activation is good or bad in AD pathogenesis is being debated, with different pieces of evidence pointing both at pathogenic and at protective aspects of microglial activation.
In the current experiments, the researchers sought to discover whether manipulating CD40L could affect Aβ levels or amyloid pathology in transgenic models of AD. They first crossed TgAPPswe mice (which carry the Swedish APP mutation and overproduce the toxic forms Aβ40 and Aβ42) and mice deficient in CD40L. These mice showed significantly reduced Aβ levels and amyloid plaque burden, as well as decreased gliosis and astrocytosis.
In a second set of experiments, the researchers showed that they could achieve the same effect pharmacologically. Mice transgenic for both the Swedish mutation and the M146L presenilin 1 mutation (PSAPP mice) were treated with an anti-CD40L antibody, with a resulting marked reduction in amyloid plaque burden and in gliosis. This was associated with an increase in circulating Aβ levels and a shift in APP processing from amyloidogenic to nonamyloidogenic fragments.
On Wednesday at the Neuroscience Conference, J.T. Roach from Mullan’s group will report in a talk (722.2) on behavioral deficits in PDAPP mice improving somewhat when treated with an antibody to CD40 ligand, and on Thursday, Jun Tan is presenting a poster on the role of the CD40 signaling pathway on neuronal differentiation and survival.
This last finding suggests that CD40L could act directly on neuronal APP processing, a possibility that the researchers explored further in neuroblastoma cells expressing human wild-type APP. In this in vitro system, the addition of CD40L shifted APP processing away from nonamyloidogenic α-C-terminal fragments and toward amyloidogenic β-C-terminal fragments. The addition of anti-CD40L antibody negated this shift.
"The main point of this paper is that the CD40-CD40 ligand interaction promotes the pathological hallmark of Alzheimer's disease, the β-amyloid plaque. The potential importance of this work is that it opens up a novel therapeutic approach for Alzheimer's disease, aimed at blocking this pathway," said author Tan.
Senior author Mullan added another emphasis to the work: "It places not just inflammation, but the immune response per se, as central to Alzheimer's disease pathogenesis."—Hakon Heimer and Gabrielle Strobel
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- Wesemann DR, Dong Y, O'Keefe GM, Nguyen VT, Benveniste EN. Suppressor of cytokine signaling 1 inhibits cytokine induction of CD40 expression in macrophages. J Immunol. 2002 Sep 1;169(5):2354-60. PubMed.
Orlando: Eyeballing the Eye in the Hunt for That Elusive Prize, an AD Biomarker
Today at the annual meeting of the Society for Neuroscience, Lee Goldstein of Massachusetts General Hospital presented evidence suggesting that the Aβ peptide occurs in the eyes of people with Alzheimer’s disease, raising the intriguing, if distant, prospect that this finding could possibly be developed into a biomarker or diagnostic test. The work is in its early stages and has not been independently reproduced.
Using a number of different antibodies, Goldstein et al. claimed to have detected Aβ in human lens tissue at levels similar to those found in brain and CSF. The scientists obtained postmortem lenses from 10 people with pathologically confirmed Alzheimer’s and reported that they found Aβ in all of them, but not in control lenses from healthy people, or people with other neurodegenerative diseases. The AD cases had Aβ levels four times higher than the controls, Goldstein said.
Goldstein cited several lines of evidence, including biochemistry and electronmicroscopic immunogold labeling of the lenses, that suggest this Aβ might occur in the cytoplasm of the lens. That would make this study the first to demonstrate cytoplasmic Aβ. Generated from APP by cleavage in a membrane compartment, Aβ has indeed been detected intracellularly (learn more about this during our upcoming Alzforum Live Discussion on intracellular Aβ with Gunnar Gouras), but to date, intracellular Aβ always appeared to be in a membranous organelle.
In studying the AD lenses, Goldstein, who works with Ashley Bush and others at MGH, discovered that the people with AD had had a form of cataract, whose incidence and prevalence rates are unknown. (Incidentally, Goldstein added, people with Down’s syndrome not only develop Alzheimer’s pathology, they almost always also develop cataracts.) A leading form of age-related blindness, cataracts come in many different forms. The one at play here, called a supranuclear cataract, is unusual in that the deposits form on the back of the lens and are not visible with the naked eye. People with this type of cataract see normally and, therefore, do not see a doctor about it, leading Goldstein to suspect that this form of cataract may frequently go undetected, even though it can be seen with the split lamps used in eye exams.
The affected cells are also unusual in that they have lost most organelles. They are largely empty sacks of cytoplasm, presumably to enable light to pass through the lens without diffraction, Goldstein said. The investigators do not know how early in the disease this change occurs. All patients studied to date had advanced Alzheimer’s. To find out, they are collaborating with Marilyn Albert and others at MGH who are following with repeated imaging tests a cohort of people to learn which measurements will enable them to predict who will convert to Alzheimer’s disease among those with mild cognitive problems.
Goldstein presented experimental data to bolster his finding, including SELDI-mass spectrometry analysis of Aβ extracted from the lens of a woman with AD, Western blots used with different sets of Aβ-antibodies, Congo Red labeling of lens tissue, and in vitro studies showing that the lens Aβ binds specifically with other cytosolic proteins, for example, the chaperone alpha-B crystalline. Other in-vitro studies indicated that lens Aβ aggregates into protofibrils in a metal-dependent oxidative mechanism, and that metal chelators can inhibit this process. The human lens contains many types of misfolded, aggregating protein.—Gabrielle Strobel
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Orlando: It’s Not Acupuncture—LTP Is Going Parallel to Figure out Early Changes in AD
In the field’s current focus to understand what might go wrong at synapses early on in AD, measuring long-term potentiation has become a widely applied tool. This phenomenon of synaptic strengthening in response to intense stimulation via NMDA receptors is thought to underlie learning and memory. The jury on that question is still out, but LTP and related measurements have become widely accepted as a sensitive method of characterizing changes in synaptic function. Consequently, a growing number of labs have imported this technique into their study of AD mouse models.
Natasha Shinsky and colleagues at Elan Pharmaceuticals in South San Francisco yesterday presented a technical twist on this approach by using a microelectrode array developed by a German company to record simultaneously from 60 channels placed all over the mouse hippocampus. While this does not in itself improve the information one receives from each recorded field, the simultaneous data gathered from multiple points can be analyzed to better understand the response from the whole system rather than from just the one spot surrounding the recording electrode.
Shinksy, with Karen Chen and others, studied PDAPP mice at two time points: at five to seven months, when they have not deposited plaques yet, and at 18 to 20 months, when plaques litter the mice’s brains. Much previous work on mice models has focused on neuronal loss (i.e., its absence) and degeneration. Recent work, in Lennart Mucke’s, Frank LaFerla’s, and Dominic Walsh’s labs, for example, hints at LTP changes prior to plaque deposition. Yet, it is not clear how that relates to cognitive deficits (see, for example, Dewachter et al., 2002).
Shinksy measured basic synaptic transmission, paired-pulse ratio, and LTP induced in two different ways. Slices of five-month-old mice showed significant impairment in only one of these measures, namely LTP induced with high-frequency stimulation. The old mice showed alterations in all parameters measured. Taken together, this suggests that PDAPP mice have problems controlling transmitter release, probably develop internal calcium overload as a result of disturbed calcium homeostasis, have abnormal inhibition by the neurotransmitter GABA, and fewer synaptic sites than nontransgenic mice, the authors propose.—Gabrielle Strobel
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- Synapses Sizzle in Limelight of Symposium Preceding Neuroscience Conference, Orlando: Day 2
- Earliest Amyloid Aggregates Fingered As Culprits, Disrupt Synapse Function in Rats
Paper Citations
- Dewachter I, Reversé D, Caluwaerts N, Ris L, Kuipéri C, Van den Haute C, Spittaels K, Umans L, Serneels L, Thiry E, Moechars D, Mercken M, Godaux E, Van Leuven F. Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci. 2002 May 1;22(9):3445-53. PubMed.
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Orlando: It’s Getting Hot around CDK5, Has the Field Noticed Yet?
In a sparsely attended slide presentation yesterday at the Neuroscience meeting, Inez Vincent of the University of Washington, Seattle, presented a feat no one seems to have pulled off before: With an experimental small-molecule compound that inhibits the kinase cdk5, she almost completely reversed neurodegeneration in a mouse model that may be obscure, but whose neurofibrillary pathology is very similar to that seen in human AD.
Vincent studied a natural mouse model of Niemann-Pick Type C (NPC). This human disease was so named because of its lysosomal storage defect, which causes ballooning neurons filled with lipid, spheroid structures in axons, and neuronal loss. Trying to understand how this pathology comes about, Vincent studied phosphorylation of cytoskeletal proteins from human disease and the mouse model. She found that tau was hyperphosphorylated in both human and mouse, and correlated with increased cdk5 activity and conversion of p35 to p25. This started at one month of age in mice. P25 and cdk5 accumulated together with hyperphosphorylated cytoskeletal proteins in axon spheroids.
The kicker, however, lies in the treatment experiment. Vincent treated four- to six-week-old mice with the cdk5/p25 inhibitor roscovitine, originally developed by a French group. The mice received intraventricular infusion driven by an osmotic pump for two weeks. Not only did the treated mice lose less weight and improve their locomotor scores compared to controls, but their protein phosphorylation decreased by about 90 percent in immunoblot, ELISA, and immunohistochemistry assays. The axonal spheroids partly resolved and neuronal loss decreased markedly. The authors conclude that cdk5/p25 mediates Niemann-Pick C neuropathology, and that inhibitors of this enzyme may treat this rare disease. The obvious implication is whether this cdk5/p25 mechanism is at work in AD, which also features tau hyperphosphorylation and neuronal degeneration. This apparently has not been tested. The drug could be tried in tau transgenic models, as well as in combined tau-APP transgenic models, which feature a more severe, combined phenotype.
Proteins Behaving Badly: P25/CDK5 Consorts with Pathologic Substrates.
Today, J.C. Cruz in Li-Huei Tsai’s lab presented an extension of Tsai’s presentation on Sunday (see related news story), in which she had suggested that hyperphosphorylation of APP’s cytoplasmic domain by p25/cdk5 influences the sorting of APP away from α-cleavage in the cell membrane and instead toward BACE cleavage in endosomes (see related news story). To examine this question in vivo, Crux et al. generated inducible transgenic mice that express p25-GFP under the control of the CamKinase2 promoter, so that the transgene is expressed only in the forebrain. With a panel of different phospho-specific antibodies, Crux found that with the induced p25, cdk5 no longer phosphorylated its normal targets, for example, the postsynaptic protein PSD-95. Instead, cdk5 now hyperphosphorylated APP, tau, and neurofilament. The authors suggest that the two regulatory proteins-p35 and p25-direct cdk5 to different sets of targets, one normal, one pathological.
The idea connecting all these dots, speculates Tsai, would be that p25/cdk5 phosphorylates tau, leading to axonal transport blockages and later to tangle deposition, and also phosphorylates APP, leading to Aβ generation in endosomes. How is that evil p25 made? The enzyme calpain, which has been implicated in neuronal death, cleaves p35 to generate p25 (see related news story). So what activates calpain? This is where the argument becomes circular, as Aβ 42 has been shown to activate. However, other toxic conditions do, as well, for example, oxidative stress and increased intracellular calcium. This ion has long been a suspect in AD, and recent studies indicate that presenilin-1 mutations, besides revving up Aβ generation, also disrupt the restorative flows of calcium between the inside and the outside of the cell in such a way that intracellular calcium rises to dangerous levels. Perhaps one way in which PS1 mutations might induce tau pathology is via this connection?
Does this simplified speculation hold water in humans? The p25-transgenic mice do exhibit neurodegeneration, and yet, does the overexpression of p25 make this an artificial, irrelevant finding? Tsai says that a mouse overexpressing an endogenous mouse protein may not be more artificial than a mouse overexpressing a human protein. Her original finding that p25 protein levels were elevated in the brains of AD patients (see news) did not address whether that varied between regions. Today, B.A. Samuels, and others in Tsai’s group report a follow-up study on another series of human autopsy samples from multiple brain regions. Working with Yong Shen of the Sun Health Research Institute in Sun City, Arizona, Samuels found that frontal cortex shows the strongest overproduction of p25 relative to p35.-Gabrielle Strobel.
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Orlando: ADDLing Pieces to the Amyloid Puzzle; Oligomers Increase in AD Brain
In three posters on Wednesday here at the annual Neuroscience meeting, William Klein’s laboratory extended their earlier presentation (see related news story) to add new pieces to the puzzle of the amyloid cascade hypothesis. One study indicates a steep elevation of the neurotoxic ligands in AD brain.
ADDLs, or Aβ oligomers, are notoriously difficult to detect in animals. Yuesong Gong presented data showing that soluble, nondenaturing extracts of AD brain tissue contained molecules that matched synthetic ADDLs with respect to size, isoelectric point, ligand specificity, and cross-reactivity. On average, these molecules were 1,200 percent more abundant in AD patients than in age-matched controls, with individual subjects elevated as much as 70-fold, the authors write.
Klein noted that the presence of oligomers in the brain is not well-recognized despite their recently verified role in interfering with LTP and the recognition that they are the putative target of therapeutic antibodies that reverse cognitive decline in mice models. "These are the hidden toxins, the missing links in the Alzheimer’s cascade," he said.
Pascale Lacor showed data that suggest a possible mechanism for how ADDLs might interfere with synaptic plasticity. Using immunofluorescent staining for synaptophysin, PSD-95, and spinophilin, the lab illustrated that ADDLs bind to receptors at postsynaptic terminals and subtly alter the structure of the terminals, including increasing the size of synaptic boutons. "ADDLs cause the synapses to show a little explosive growth, and this probably interferes with information storage," says Klein. For more on synaptic changes in AD, see related news story and related news story.
In a third poster, Lei Chang described an immunoassay which the lab developed to screen for molecules that inhibit ADDL formation. After screening several beta cyclodextrin-based libraries, the group identified one that was effective in blocking ADDL formation and toxicity at micromolar doses. They are now working to refine the compounds.
"The implication is that the way to help people with AD is to get rid of these molecules," said Klein. He believes oligomers present a better target for therapeutics and antibodies than do fibrils or plaques, not only because the impact of these soluble ligands is associated with synapse interference, but also because of the inflammatory reaction seen with the vaccine therapy developed so far. "If you have the antibodies bind to soluble molecules, you can avoid that inflammation," said Klein. To this end, Klein’s team has generated antibodies against ADDLs that target their assembled forms. The challenge ahead is to find antibodies that bind more specifically to ADDLs, but not to fibrils, in the hope of affecting a discrete pathway.—By guest writer Brenda Patoine.
Brenda Patoine is a science writer in Lagrangeville, New York, who writes for BrainWork and other publications.
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Orlando: ARF Awards 2002
It may not be the Oscars or Nobel prize, but recipients of the second annual Alzheimer Research Forum Awards were warmly applauded during the Society for Neuroscience meeting's Alzheimer Disease Social. The Innovation Award was given to Inez Vincent for her authoritative review of cell cycle genes in AD (see "The Cell Cycle and AD-Let's Unite for Division"). Outstanding Contributor Awards were given to Todd Golde, David Holtzman and Dominic Walsh. A special category, the Mensch Award, was created to honor two original members of the scientific advisor board, Paul Coleman and Peter Davies, who have remained prolific contributors to the Alzheimer Research Forum.
Following the presentation of the awards, the audience was treated to a series of comic sketches orchestrated by Ben Wolozin. Ben, Rachael Neve, Rudy Tanzi, Dennis Selkoe and Jeff Nye enacted a parody of an NIH study section reviewing grants such as a $1 billion request submitted by the Medellin cocaine cartel to study "APP Trafficking." Next came a skit featuring Alzheimer Research Forum Editor June Kinoshita impersonating Dora Games, interviewing Mucke Mouse (Mickey's scientist cousin), played by Gina Zainelli, an intrepid graduate student. Rudy Tanzi masterfully improvised the musical sound-effects. Jeff Nye followed with a talk explaining the pharma approach to AD research. Ben Wolozin wrapped up with a Saturday Night Live-style "Week in Review," which was accompanied by tasteful slides such as John Trojanowski and Virginia Lee garbed in spandex Spiderman and Cat Woman outfits; Saddam Hussein posing for a photograph with mustachioed lookalikes, Rudy Tanzi and Sam Sisodia; and Khalid Iqbal and Dennis Selkoe with the heads of their scientific competitors grafted to their shoulders.-June Kinoshita.
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Orlando: Preliminary Results of NGF Gene Therapy Trial Are Mixed
Preliminary results of the NGF gene therapy clinical trial in early-stage AD illustrate the potential promise-and pitfalls-of gene therapy for neurodegenerative diseases.
Mark Tuszynski of the University of California, San Diego, presented early data from this first clinical use of gene therapy in humans with AD at the Neuroscience meeting last Wednesday. Alzforum covered the start of this trial (see related news story). To date, eight patients have received transplants, which are autologous grafts of skin fibroblasts transfected with the human NGF gene. Two of the eight developed serious brain hemorrhages due to movement during surgery, and one died as a result.
Surgical techniques for subsequent patients were then refined, Tuszynski said. While the procedure is not complex, he noted that it is technically challenging due to the careful planning that is required to insert a needle deep into the brain without passing through sulci or gyri, which could cause hemorrhaging. The target of the grafts is very specific: It is the nucleus basalis of Meynert, a basal forebrain area that sends cholinergic projections to the hippocampus and cortex. Specially designed needles and anesthesia protocols have been developed for the surgeries, and stereotaxis is used to stabilize the head during the procedure. Whereas early patients received only mild anesthesia—the thinking being that keeping the patient cognizant during surgery would be beneficial—patients are now deeply anesthetized during the surgery to ensure that they do not move.
The eighth and final patient in this Phase I safety study received the NGF implant on November 1. Each pair of patients has received a progressively greater volume of transplanted cells, and the researchers waited two months in between each patient’s surgery. The first two subjects received grafts of a small volume of cells to one side of the brain, while the next two received the same cell volume bilaterally. The fifth and sixth patients received double the volume, and the final two got five times the initial dose. Five women and three men, age 53 to 76, are enrolled.
The team is now following the six patients in whom the surgery did not produce complications. No adverse effects on cognition have been seen, which Tuszynski said is an indication that the genes and vector are safe. The grafts are morphologically similar to those in the primate models on which this trial was based. It is too early to draw any conclusions regarding efficacy. Tuszynski explained that there is evidence the grafts have been innervated by cholinergic axons and presented MRI scans from one patient that indicated a greater uptake of cholinesterase on the side where cells were implanted. The full data set is expected in one year.
Tuszynski concluded that the study indicates that cholinergic neurons can be accurately targeted for NGF grafts, that implanted cells express NGF without adverse effects, and that patients need to be deeply anesthetized. If the pilot is successful, a follow-up trial will likely utilize an in-vivo gene transfer technique (as opposed to this ex-vivo protocol), which was not adequately developed at the start of this trial, but is hypothetically safer because it requires fewer needle passes into the brain. Systems where NGF expression can be regulated are "not yet ready for clinical trial use until immunological issues are sorted out," Tuszynski said.—By guest writer Brenda Patoine.
Brenda Patoine is a science writer in Lagrangeville, New York, who writes for BrainWork and other publications.
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Orlando: Medium-Throughput Target Discovery Using Brain Slices
Researchers led by Don Lo of Duke University Medical Center and Cogent Neuroscience, Inc., in Durham, North Carolina, have developed a novel brain-slice platform for identifying therapeutic targets for neurodegenerative diseases, including Alzheimer’s, Huntington’s, and Parkinson’s, as well as stroke and glaucoma. At this month’s Neuroscience meeting, Lo’s group presented eight posters describing their various models and preliminary results of "hits" they have identified so far.
The platform is based on rodent brain slices that are biolistically transfected, that is, with a gene gun that employs a pulse of helium to fire small gold particles coated with human DNA and fluorescent reporter constructs at living brain slices, which are kept viable in culture for up to two weeks. The tissue slices retain their three-dimensional structure, enabling testing on intact living brain cells in a quasi-natural environment including surrounding glial cells.
Lo called the model "almost in vivo, essentially an animal model in a dish. We’ve used every trick in the book to recapitulate the neurologic diseases we’re interested in studying-genes, environmental insults, whatever we know causes the disorder in humans." The team presented data in Orlando on their models for stroke, glaucoma and Huntington’s disease. The effort attempts to overcome the time constraints of using transgenic animal models to test drug candidates and the high failure rate of compounds identified in cell-free protein-binding models. Lo also sees it as helping to close the "innovation gap" between academic basic research, where compounds may be discovered, and the high-throughput screening that typically occurs only at biotech and pharmaceutical firms.
The job of identifying therapeutic targets is not driven by hypotheses, Lo said: "We just throw drugs and disease genes at these models and see what blocks the pathology." So far, his group has identified some new drug candidates that have not yet been investigated for therapeutic use. They also have shown that their assays could have predicted the failure of drugs that did not show efficacy in clinical trials, including a number of the neuroprotective drugs that were investigated for stroke. While the brain-slice test confirmed that these compounds were indeed neuroprotective, the protection was seen only within a narrow set of experimental conditions, which may explain their clinical disappointment.
Once a disease model is developed, the team can screen about 1,000 genes or compounds in a matter of months, said Lo’s colleague Peter Reinhart, also at Duke and Cogent. To date, they have screened 5,000 molecules against the stroke model, and a few hundred genes and 3,000 compounds using the Huntington’s model. The method enables investigation of multigenic diseases; Reinhart said they have used as many as 11 genes in one model. The team is just beginning to screen compounds for Alzheimer’s using a model developed with combined DNA constructs for APP, tau, Aβ42 and ApoE4. (The Alzheimer’s model was not presented.)—By guest writer Brenda Patoine.
Brenda Patoine is a science writer in Lagrangeville, New York, who writes for BrainWork and other publications.
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