Linking calcium, oxidative stress, and the role of DJ-1 in Parkinson's disease

PD is a neurodegenerative disease whose defining pathology is the selective degeneration of dopamine producing neurons in an area of the midbrain called the substantia nigra. The biggest mystery in PD research has centered around why these particular neurons degenerate. Research now offers a clue and a tantalizing drug target that may help in treating PD patients. A team led by Dr. James Surmeier, a professor at Northwestern University, has shown that a specific type of calcium channel expressed in substantia nigra dopaminergic neurons may render them more vulnerable to damage by oxidative stress (Guzman et al, 2010). By targeting this channel scientists may be able to develop an entirely new avenue of therapy for PD that would prevent the associated neurodegeneration. The best pharmacological treatment currently prescribed for Parkinson’s disease (PD), the dopamine precursor L-DOPA, was discovered over forty years ago. At best, the administration of L-DOPA can lead to a temporary reprieve from the debilitating motor symptoms associated with PD, but it does not affect disease progression and patients eventually develop a tolerance to it. L-DOPA is still the most widely prescribed medication for PD due to lack of a disease modifying therapy. Dr. Surmeier’s group now offers hope for a new therapy that could block the influx of calcium into dopaminergic (DA) neurons and prevent the progression of PD.

It had been shown previously by the same group that the activity of Cav1.3 (L-type) calcium channels, which allow calcium to enter the cytoplasm of the cell, was not necessary for the functioning of DA neurons, specifically for their pacemaking activity (Guzman et al, 2009). Furthermore, blocking these channels with a chemical called isradipine led to a rescue of DA neuron loss in a neurotoxin mouse model of PD (Chan et al, 2007). So what is it about these channels that is so bad for these highly active DA neurons?

To answer this question, the lead author of the paper, Jamie Guzman, created transgenic mice expressing the redox-sensitive version of GFP (roGFP) under the TH promoter. Upon getting oxidized, roGFP shifts it 490/400 nm excitation spectrum and can be differentiated from non-oxidized roGFP. Furthermore, the construct contained a matrix targeting sequence designed to localize the protein to the mitochondria, which have been centrally implicated in mechanisms of PD pathology. The team then used these mito-roGFP transgenic mice to measure the level of oxidation present in DA neurons from their brains.

Guzman et al found that substantia nigra (SN) DA neurons exhibit a much higher basal level of cellular oxidative stress than DA neurons from the neighboring ventral tegmental area (VTA) which lack these L-type calcium channels. This suggested that Ca2+ influx may somehow be responsible for increased levels of oxidative stress. Indeed when they blocked L-type channels with isradipine, the level of oxidative stress decreased dramatically in SN DA neurons. The research group went on to show that a curious phenomenon, mitochondrial uncoupling, occurs in SN DA neurons as a compensatory mechanism against increased levels of oxidative stress. By uncoupling the mitochondrial electron transport chain from the production of ATP, these neurons are able to reduce the production of toxic reactive oxygen species (ROS) which are a byproduct of the respiratory chain. The authors demonstrated that the uncoupling events, measured by fluctuations in the mitochondrial membrane potential, were dependent on Ca2+ influx and levels of ROS. Again, blocking L-type calcium channels decreased the incidence of these uncoupling events, presumably by reducing oxidative stress levels in the neurons. The data suggests that Ca2+ influx, oxidative stress, and mitochondrial uncoupling are intricately linked together.

The most interesting part of this study was the connection that the authors found between these processes and DJ-1, a protein implicated in Parkinson’s. People with a homozygous loss-of-function DJ-1 mutant genotype develop an early-onset form of PD. To test the role of DJ-1 Guzman et al used a mouse with the DJ-1 gene knocked out. DJ-1 knockout DA neurons exhibited very low levels of mitochondrial uncoupling and correspondingly higher levels of oxidative stress, suggesting DJ-1 might somehow be regulating the mitochondrial response to oxidative stress. Amazingly, blocking the L-type calcium channels completely rescued this oxidative stress effect. Earlier work had implicated DJ-1 in redox signaling pathways and upregulation of antioxidant proteins (Kahle et al, 2009). Thus the authors decided to check whether DJ-1 was exerting it’s effects at the gene expression level. They observed that DJ-1 knockouts had lower transcript levels of some of the key mitochondrial uncoupling proteins, but expression of antioxidant enzymes was unaffected. The authors concluded that a loss of DJ-1 functionality weakened the compensatory mechanisms in mitochondria, making the DA neuron much more vulnerable to oxidative stress in the SN. This may explain why people with homozygous DJ-1 mutations end up suffering from early-onset PD.

The key finding of this study was that the intracellular impairments resulting from a DJ-1 mutation can be reversed by using L-type calcium channel blockers such as isradipine. Isradipine belongs to a class of molecules known as dihydropyridines which are widely prescribed as a treatment for high blood pressure in humans. Perhaps most importantly, these drugs can cross the blood-brain barrier. Recent epidemiological studies support a decreased risk of developing PD in chronic users of dihydropyridines (Becker et al, 2008; Ritz et al, 2010). This offers a ray of hope for PD patients. In fact, clinical trials using isradipine in PD patients have already begun and are in Phase II, with Phase III trials expected to be launched in the next year.

The research by Dr. Surmeier’s group is remarkable in many ways in that it not only elucidated the mechanism underlying the function of a protein implicated in PD, but also identified a drug target and a potential drug candidate that is already in clinical trials. However a lot more needs to be understood about L-type calcium channels and their role in genetic as well as sporadic forms of PD. It may well be that blocking these channels only offers protection in DJ1 knockout and neurotoxin models of PD. Lets keep our fingers crossed that this is not the case.

A link to the Guzman et al study can be found here: http://www.nature.com/nature/journal/v468/n7324/full/nature09536.html

[Mice] Singing in the Lab

Happy New Year everyone! To celebrate the start of 2011, here is a story from the end of last year: "singing" mice.

These mice, generated by a team of Japanese scientists, vocalize in a manner startlingly similar to that of songbirds. These mice are the product of the University of Osaka's Evolved Mouse Project, which screens mice prone to mis-copying DNA for the presence of random mutations. So far, there are no details as to what exact mutation resulted in these mice exhibiting the song-like vocalizations.

For video of the mice, see this Youtube video.

Interestingly, it appears that non-mutant mice do vocalize extensively, just at ultrasonic frequencies (see this video detailing the research of Dr. Christine Portfors). Could the mutation in the Osaka mice be affecting the frequency of mouse vocalizations, reducing them to levels discernible by human ears? Any commentators out there with greater familiarity in songbird and mouse vocalizations, please sing out in the comments.

Stanford Journal Club: Common Ancestry for Mushroom Bodies and the Cortex?

Part of the Stanford Neurosciences curriculum is attendance at a weekly journal club wherein students take turns presenting 30 minute presentations on current or classic papers. Recently, Poh Hui Chia, a 3rd year graduate student in the laboratory of Kang Shen, presented a paper recently published in Cell that used a novel computational technique to examine the possibility that mushroom bodies and vertebrate pallium (aka cortex) could have shared a common ancestor.

The Paper

Profiling by Image Registration Reveals Common Origin of Annelid Mushroom Bodies and Vertebrate Pallium, by Tomer et al, contains a methodologically complex analysis of key cellular and molecular features of both the marine annelid worm, Platynereis' mushroom bodies and the mouse pallium. Those readers with an appreciation for comparative neuroanatomy are sure to enjoy Poh Hui's discussion of the paper's unique computational methods and intriguing results.

In the introduction to their paper, Tomer et al describe the highlights of their paper as follows:

► A new protocol for cellular resolution expression profiling by image registration ► Generation of a multigene map of the developing annelid brain ► The annelid mushroom bodies and the vertebrate pallium share molecular coordinates ► Homology of sensory associative brain centers in Bilateria

JC Presentation Part 1

JC Presentation Part 2

Abstract

From Tomer et al, 2010:

The evolution of the highest-order human brain center, the “pallium” or “cortex,” remains enigmatic. To elucidate its origins, we set out to identify related brain parts in phylogenetically distant animals, to then unravel common aspects in cellular composition and molecular architecture. Here, we compare vertebrate pallium development to that of the mushroom bodies, sensory-associative brain centers, in an annelid. Using a newly developed protocol for cellular profiling by image registration (PrImR), we obtain a high-resolution gene expression map for the developing annelid brain. Comparison to the vertebrate pallium reveals that the annelid mushroom bodies develop from similar molecular coordinates within a conserved overall molecular brain topology and that their development involves conserved patterning mechanisms and produces conserved neuron types that existed already in the protostome-deuterostome ancestors. These data indicate deep homology of pallium and mushroom bodies and date back the origin of higher brain centers to prebilaterian times.

Profiling by Image Registration Reveals Common Origin of Annelid Mushroom Bodies and Vertebrate Pallium. Tomer et al (2010). Cell 142(5): 800-809.

U Can't Touch This

Another epic moment in PubMed searches, presented without comment. [Courtesy of Stanford graduate student Geeorge Sebastià Vidal Pérez-Treviño.]

SFN: Robert Wurtz's Lecture - A perspective

The neural basis of cognitive phenomena: are monkeys the ideal model?Sridhar Devarajan

In his presidential special lecture delivered to the Society for Neuroscience (Nov.15, 2010), Prof. Robert Wurtz highlighted the importance of non-human primates (monkeys) in cognitive neuroscience research. Monkeys can be trained on a variety of complex behaviors, and exhibit brain structures that are remarkably homologous to the human brain. While acknowledging that the choice of model organism should be driven by the research question, Prof. Wurtz underscored the special status of monkeys as the model of choice for understanding the neural basis of cognitive phenomena, such as attention.

While appealing, this argument begs the question: are such cognitive phenomena unique to primates? Take attention, for instance. Attention control in primates is known to be of two kinds: “goal-directed” and “stimulus-driven”. During the performance of a demanding task, we engage our attention on task-relevant stimuli in a “goal-directed” fashion. For instance, when driving to a friend’s place in an unfamiliar neighborhood, our attention is closely engaged in tracking street signs and other relevant landmarks, while ignoring irrelevant details of the landscape. On the other hand, unexpected and highly salient stimuli automatically draw (capture) our attention (“stimulus-driven”). For instance, a sudden, loud siren immediately draws our attention to the approaching fire truck, so we can get out of its way quickly, if need be.

How certain are we that similar kinds of attention control do not operate in the brains of other classes of (non-primate) animals? How about insects? Bees, for example, forage for nectar-bearing flowers matching a specific sensory template while carefully avoiding physically similar, but irrelevant objects. On the other hand, a sudden bright light, or a loud noise startles bees and quickly draws their (collective) attention to the source of the disturbance, as many a gardener who has accidentally disturbed a beehive could painfully attest. Hence, it is difficult to assert that similar forms of “goal-directed” and “stimulus-driven” attention control do not operate in the primitive insect brain.

Do insects share other “cognitive” phenomena besides "attention"? How about "motivation"? Symbolic communication? Learning? Memory? Perception? Where can we draw the line in the animal kingdom in terms of these phenomena? And where does this leave us with regard to the original question: Are monkeys indeed the ideal model for understanding the neural bases of cognitive phenomena?

Prof. Bob Wurtz’s assertion is relevant for cognitive neuroscientists who wish to understand the mechanistic basis of cognitive phenomena in the human brain. Indeed, such knowledge is fundamental to diagnose and treat cognitive disorders (such as schizophrenia, and autism) that afflict the human brain, and for which no cures are currently available. However, from a basic science perspective, Prof. Wurtz’s proposal leaves little room for understanding the emergent principles of brain circuits and their computations that give rise to cognitive phenomena, such as attention.

An analogy, might help illustrate this distinction. I have had the unique experience of trying to figure out the rules of American football by watching two professional teams compete. While I broadly understood that each team’s objective was to get the ball across the opponents' line at the other end of the field, the gameplay proceeded so quickly, and with such intricately complex maneuvers, that I completely failed to decipher the essential rules: the rules of passing, the concept of a “first-down” and the like. The fact that the players on the field appeared to  transform, without warning, into a completely different set of individuals did not help either. Perhaps, it would have been easier for me to figure out these essential rules, had I observed the simpler gameplay of middle schoolers less encumbered by advanced, and complex strategies.

A basic goal of cognitive neuroscience is to understand core principles by which cognitive phenomena arise in the intricate wetware of the brain. Prematurely restricting study of the neural basis of cognition to specific animal models raises the potential risk of "overfitting", of failing to unravel fundamental principles of neural circuit operation that give rise to such phenomena.

Molecular Animation: Cinematic Science

From the animators that brought us the glorious video, The Inner Life of the Cell in 2006, comes another tour de force in molecular animation. This time, the collaborative team of BioVisions, a scientific visualization program at Harvard’s Department of Molecular and Cellular Biology, and the Connecticut-based scientific animation company Xvivo have created a video entitled Powering the Cell: Mitochondria.

As with their previous video, Powering the Cell: Mitochondria is a glorious depiction of intricate cellular processes that combines detailed science with beautiful animation. Both videos can be seen below (Inner Life after the break), and the field of scientific animation can be learned about by reading the recent NYTimes article of molecular animation.

Powering the Cell: Mitochondria

The Inner Life of the Cell

SFN: Brain Romance: Larry Young and Social Bonding

First up on this last morning at SFN2010 is Larry Young from Emory who will be discussing the Neurobiology of Social Bonding and Monogamy: Implications for Autism Spectrum Disorders. To start out the talk, Young discusses examples of pair bonding and monogamy in the animal kingdom. First, some jokes regarding human monogamy, which went by two fast for me to type down. These are followed by a brief mention of prairie vole pair bonding, to which Young will return multiple times during the course of the talk. And finally, a more exotic example: monogamy in the deep sea. The deep sea angler fish live deep in oceans, and displays a rather extreme form of pair bonding. To quote Larry Young*,  “a male can spend half of his lifetime searching for a female. So when the male finds the female, he forms a bond. And not an emotional bond. A physical bond.” These extreme changes in the male’s circumstances ensure that the male WILL reproduce every time the female spawns.

Did pair bonding evolve by tweaking mechanisms that promote maternal bonding? Pair bonding has evolved multiple times, and it seems unlikely that such behavior would have evolved newly each time. So the hypothesis is that pair bonding is a tweaking of the maternal bond that is evident in many (if not all) mammals. It is known that the chemical oxytocin is highly involved in regulating the peripheral physiology of reproduction, as well as the transition to maternal behavior following successful reproduction.

An example: virgin rats avoid pups, but a day before they give birth, there is a behavioral change in that pregnant rat will start to seek out pups. Oxytocin is critical for this behavioral switch – injection of oxytocin into virgin rats will cause them to seek out pups. The same is true for sheep and bonding with lambs (except with oxytocin release stimulated via cervicovaginal stimulation of the female sheep).

And so back to pair bonding in voles. The basic behavioral assay is called the Partner Preference Test, details of this assay can be found in the literature, but, in brief, involves allowing a female and male to mate, and then presenting the male with a novel female and its mating partner. The male will spend the vast majority of its time with its mating partner, and will in fact display aggressive behavior towards the novel female. [Bloggers note: video of the pair bonded voles clearly demonstrates the complete adorability of social interactions between pair bonded animals.] Oxytocin is required for female pair bonding.

And now we move onto the prairie vole versus meadow vole story. I highly recommend reading the original papers, but the basic gist of the research is that prairie voles display social pair bonding while meadow voles do not.

Oxytocin release into nucleus accumbens causes pair bonding. It makes sense that there is a conserved oxytocin mechanism underlying both pair bonding and maternal bonding. Think back to the research showing that in female sheep, cervicovaginal stimulation (which simulates birthing) releases oxytocin and results in maternal bonding. This mechanism of oxytocin release could easily also underlie pair bonding because, to quote, “there is a heck of a lot of cervicovagina stimulation going on when females are mating with males.”

So female bonding involves release of oxytocin, but what about male pair bonding. Vasopressin plays an important role in pair bonding in male prairie voles (also underlying territorial behavior, scent marking, and aggression).  A bit of wild speculation – female pair bonding may have evolved from the maternal bond mechanisms, whereas perhaps male pair bonding evolved from the territorial processes. Another great quote (paraphrase, really): “So male voles consider females to be part of their territory. I’m only talking about prairie voles here.” Animals without of oxytocin and vasopressin have social amnesia.

Other molecules required for partner preference include dopamine and opiates, which suggests a link between mating and the classical reward circuitry in the brain. How does mating activate the reward system? During mating, the sensory stimuli activate the VTA causing release of dopamine/opiates into reward areas of the brain. Experimental evidence of a link between sex and reward learning: “we know that a male rat will press a lever to get a female rat to drop out of the ceiling”.

Why do meadow voles not form pair bonding? Interestingly, there is a lack of vasopressin receptors in the ventral pallidum in meadow voles, but not in prairie voles. Expressing vasopressin receptors in this area in meadow voles causes all of the “transgenic” voles to form pair bonding. Looking at the genetic mechanisms underlying the difference in vasopressin receptor expression, they first looked at differences between species (see previously published work for this story), but then ended up looking at individual variation within the avpr1a gene (vasopressin receptor) within the prairie voles. Interestingly, variation in the length of the microsatellite associated with the avpr1a gene can predict how likely the animal is to form a pair bond. Males with short microsatellite sequences are less likely to form a pair bond than males with long microsatellite sequences.

Is the work in voles applicable to humans? Another great quote (again, probably a paraphrase) “I work at a medical school, so I’m always asked, ‘Well these prairie voles are cute and everything, but how can we use…profit… off of this’”. Several groups are looking at human pair bonding, as well as levels of oxytocin and vasopressin in humans. On example is a recent paper that showed that genetic variations in the vasopressin receptor (again, avpr1a) predict whether human males will report a crisis in their marriage. (Bloggers note: commentary from the grad student sitting next to me: “All right, I’m getting a spit test.”). Further studies regarding the role of oxytocin in human relationships involve giving humans oxytocin, and how shown that this treatment can:  improve “mind-reading” in humans, improve the likelihood of eye contact between partners, increase emotional empathy and enhance socially reinforced learning. From these studies came the hypothesis is that oxytocin is acting by increasing the social saliency of the task stimuli, and this hypothesis brings us to the topic of autism. Autism spectrum disorder is characterized by a deficit in social interactions. Current strategies for enhancing oxytocin function involve identifying novel drug targets for promoting endogenous oxytocin release. One identified drug is melanotan II, which is also used in tanning and erectile dysfunction. Importantly, meadow voles that are given melanotan II form pair bonds, most likely because the drug stimulates endogenous release of oxytocin. This research has suggested that meadow voles might be a useful model for screening drugs for enhancing social cognition – this possibility is being explored, alongside work to develop primate models that could be used to test any drugs identified in the vole model as possibly enhancing social cognition.

And finally, a plug for Emory Universities new center for translational work.

For more information of Larry Young and his lab's current work, visit his lab website.

*I have attempted to quote verbatim from Larry Young's talk. All attempts to do so are indicated by quotations. However, given discrepancies in speed of his speech versus my typing, some quotations may not be exact.

SFN: Area PITd, a novel cortical area controlling visual attention?

I’ll admit that I listened to this talk only because it immediately preceded a talk on cholinergic modulation of visual and olfactory attention. However, I’ll be posting my notes from this talk. My reasons are two-fold: 1) the visual/olfaction talk was not as satisfying as I had hoped and 2) this talk appeared to reflect a solid scientific effort. Working on a mid-brain structure (the superior colliculus, thus the interest in attention), I am not as familiar as some about different cortical areas, and the process of identifying the involvement of those areas in particular processes. Those readers who are more familiar, please comment on your impressions of the research below. Without further ado…

This talk was entitled Cortical Area PITd, a ventral pathway area for the control of spatial visual attention?, by H. Stemman and W. Freiwald from Rockefeller University. As suggested by the title, the research identifies a role for a novel cortical region in the control of attention.

Starting off, the speaker (Stemmann) defined the functional properties that a region must exhibit in order to be classified as an attention control area. First, neurons within the region must encode visual information in a featureless manner. Second, the area should be involved in different kinds of attention. Finally, and most critically, electrical stimulation of the region must enhance the representation, and facilitate the recognition of objects in a spatially specific way.

The researchers started out using fMRI on monkeys to detect brain areas showing attention-related enhancement of activity. Their study identified an area in the inferior temporal cortex: dorsal posterior inferior temporal cortex. They note the magnitude of the attentional modulation in PITd was similar to modulation also seen in V1, V2, MT, FEF and LIP of their monkeys.

The researchers then recorded from PITd with multiunit electrodes while presenting the monkey with a variety of visual stimuli. They found no direction selectivity, but they did see enhanced firing activity when animal is directing its spatial attention to an area inside the receptive field of a recorded neuron. They recorded activity during a behavioral task, and were able to infer the trial outcome based on the spiking activity of the neuron (enhancement of firing rate was that strong). Furthermore, responses of PITd neurons accurately predicted task errors. This high degree of predictive ability was true for their entire cohort of 56 neurons.

Although they found no explicit direction selectivity, the researchers wondered whether the activity of PITd neurons was dependent stimulus motion (given that their stimulus involved fields of moving dots)? In order to confirm that the attentional effects/activity was not dependent of stimulus motion, they switched paradigms to a color discrimination task. They found that PITd neurons had no color tuning, but were still strongly modulated by attention.

From these results, they concluded that neurons located in PITd do not encode visual features of the stimulus, and therefore encode visual information in a featureless manner, and are involved in different kinds of attentional tasks (based on attentional modulation during two distinct task paradigms).

With confirmation that PITd satisfied two of the criteria for categorization as an area involved in controlling attention, the researchers next attempted to determine whether the region satisfied the third criteria. They asked whether electrical stimulation of PITd would cause changes in attention. Stimulation of PITd neurons when the visual target was inside the neurons’ receptive field result stimulated PITd when the target was inside the receptive field, saw an increase i

Electrical micro-stimulation inside receptive field of a PITd neuron made the monkey perform the visual task better. Micro-stimulation of PITd outside the receptive field caused the monkey to make significantly more errors as if attention was being directed to a spatial location outside the receptive field. Put another way, following micro-stimulation outside the receptive field, the monkey used the motion information encoded by the distracter to make their decision, not the cued location to which they should have been paying attention. These results suggested that by stimulating PITd, they could shift the spotlight of attention from to the cued location to the micro-stimulated location.

In summary, the presenter concludes that his research suggests that cortical area PITd, a ventral pathway area, may be involved in the control of visual attention.

A note that it is unclear (at least to me) what are the efferent and afferent connections to PITd, or how PITd fits into the established cortical attention circuit. However, the strength of the attentional modulation of PITd neurons, and their apparent ability to shift spatial attention is certainly impressive.

Sfn: An ode to #SFN10banter

Last night, I attended #sfn10banter, a social gathering of science bloggers and Twitter users. As promised*, a poetic celebration of the event:

The tweeters said "Hey we should meet up!" "Get some drinks, shoot the shit, put our feet up." We came at a canter, For SFNbanter. Great to meet everyone at the tweet-up.

*@Tideliar: as promised, official, poetic coverage