Diet Restriction Slows Neurodegeneration and Extends Lifespan of DNA-Repair-Deficient Mice

DNA damage occurs in human cells at a constant rate. These cells are usually able to repair themselves, but sometimes deficiencies in certain genes cause the repair process to shut down. When damaged DNA isn’t fixed, mutations can occur that cause accelerated aging or cancerous tumors to form (Hoeijmakers, 2009). Scientists at Erasmus University Medical Center in Rotterdam have found a way to slow down the process – at least in mice.

In a study published in Nature, the researchers report that when mice deficient in the DNA-repair genes Ercc1 or Xpg are put on a restricted diet, they experience better overall health and increased lifespans compared to DNA-repair-deficient mice fed a normal diet. They also found significantly lower levels of neurodegeneration in the brains and spinal cords of diet restricted animals compared to controls.

“Here we report that a dietary restriction of 30 percent tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated aging Mice undergoing dietary restriction retained 50 percent more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum,” (Vermeij, et al 2016).

Since the DNA-repair-deficient mice were already smaller and weaker than normal mice, the Rotterdam researchers wondered whether diet restriction would be beneficial or detrimental to their health. They found that gradually restricting the diets of DNA-repair-deficient mice starting at age seven weeks increased their median lifespans from 10 to 35 weeks in males and 13 to 39 weeks in females as compared to controls.

They also saw significant differences in the levels of neurodegeneration between these two populations. Using Stereo Investigator, they found 50 percent more neurons in the brains of diet-restricted mice compared to those fed a normal diet. They also saw lower levels of cells expressing p53 – a protein expressed in response to DNA damage – in diet-restricted mice.

According to the authors, dietary restriction may not fix defects in DNA repair mechanisms, but it may help to reduce the severity and speed at which the damage occurs.

“Our findings establish the Ercc1 mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general,” (Vermeij, et al 2016).

Vermeij W.P., Dollé M.E.T., Reiling E., Jaarsma D., Payan-Gomez C, Bombardieri C.R., Wu H., Roks A.J.M., Botter S.M., van der Eerden B.C., Youssef S.A., Kuiper R.V., Nagarajah B., van Oostrom C.T., Brandt R.M.C., Barnhoorn S., Imholz S., Pennings J.L.A., de Bruin A., Gyenis Á., Pothof J, Vijg J, van Steeg H., and Hoeijmakers J.H.J. (2016) Restricted diet delays accelerated aging and genomic stress in DNA repair deficient mice. Nature 537, 427-431, doi:10.1038/nature19329

Hoeijmakers JH (2009) DNA Damage, aging, and cancer. N Engl J Med; 361:1475-1485, DOI: 10.1056/NEJMra0804615

Stock image of DNA used in accordance with the CC0 public domain license.

A complete guide to imaging and analyzing spines and neurons with Neurolucida 360


Following a well-designed protocol is essential to achieving accurate and consistent results in scientific research. Now, scientists using Neurolucida 360 for dendritic spine and neuron analysis can follow a published set of guidelines to ensure optimal confocal data series for proper dendritic spine quantification and neuron reconstruction. The paper, written by MBF Bioscience scientists and researchers from the Icahn School of Medicine at Mount Sinai in New York, was published in Current Protocols in Neuroscience.

The four protocols describe best practices for imaging and analyzing dendritic spines and entire neurons. Clearly laid out procedures specify necessary materials, image acquisition techniques, and analysis procedures with Neurolucida 360.

Imaging technique is crucial to obtaining unbiased, reproducible results. Clear, crisp images captured with an appropriate z-interval will make analysis with Neurolucida 360 easier and more accurate. Throughout the paper, the authors emphasize the importance of image scaling parameters and unbiased sampling for achieving repeatable results. They also discuss the benefits of correcting optical distortion, especially in the Z-plane, with deconvolution to acquire clear images – a process critical to getting the most accurate representation of dendrites and spines.

Dendritic spine analysis is traditionally performed through tedious, time-consuming manual techniques. According to the paper, this has spawned a growing interest in a more efficient solution for spine quantification and morphological analysis like the one Neurolucida 360 provides. A software platform for automatic neuron reconstruction and spine detection in a 3D environment, Neurolucida 360 offers a variety of benefits, including:

 

  • Fast and accurate spine detection and neuron reconstruction
  • Accurate spine classification and length quantification using a five-point segment that more accurately models the spine backbone.
  • 3 user-guided and automatic algorithms to accurately model neurons visualized with multiple methodologies and imaging techniques.
  • A large number of metrics, including volume, length, and surface area.

 

“We believe that the new quantitative software package, Neurolucida 360, provides the neuroscience research community with the ability to perform higher throughput automated 3D quantitative light microscopy spine analysis under standardized conditions to accelerate the characterization of dendritic spines with greater objectivity and reliability,” (Dickstein, et al. 2016)

The full paper can be found here.

An infographic quickly outlines Protocol 1: Imaging of fluorescently labeled dendritic segments. Use this as a quick reference tool in your lab (right-click on it to save as an image):

Dickstein, D.L., Dickstein, D.R., Janssen, W.G.M., Hof, P.R., Glaser, J.R., Rodriguez, A., O’Connor, N., Angstman, P., and Tappan, S.J. 2016. Automatic dendritic spine quantification from confocal data with Neurolucida 360. Curr. Protoc. Neurosci. 77:1.27.1-1.27.21. doi: 10.1002/cpns.16

Uncovering the role of microglia in fetal alcohol spectrum disorders

microglia_alcohol

Representative images of Iba-1+ microglia in the postnatal day 10 rat hippocampus. Image courtesy of Anna Klintsova, PhD.

Children born with fetal alcohol spectrum disorders face a range of physical and cognitive impairments including long-term deficits in learning, behavior, and immune function. In a paper published in Neuroscience, Dr. Anna Klintsova and her lab at the University of Delaware report that activation of the brain’s immune response may contribute to some of the damage caused by fetal alcohol spectrum disorders.

In their study, the researchers used Stereo Investigator and Neurolucida to examine the hypothesis that exposure to alcohol while the brain is growing rapidly is associated with abnormal microglial activation and high levels of pro-inflammatory proteins which impair learning-related plasticity; leading to neuro-developmental and psychopathological disorders.

“My lab has been using both Stereo Investigator and Neurolucida for more than a decade in all quantitative neuroanatomical studies, including the featured one,” said Dr. Anna Klintsova. “We find this software to be user-friendly, reliable and essential for obtaining unbiased results.”

They used Stereo Investigator to quantify the number of microglia in the hippocampus of neonatal rats who were exposed to alcohol during the equivalent of the third trimester of a human pregnancy. The researchers expected to see an increased number of microglia in alcohol-exposed neonatal rats, however they found a decreased number of microglia. Despite the decrease in microglia number, there was a significant increase in pro-inflammatory proteins expressed by microglia and an increase in microglial activation.

To measure microglial activation, the researchers quantified the area of cell territory using Neurolucida. Activated microglia have a smaller cell territory than resting microglia, so the smaller cell territory found in alcohol exposed rats indicates a more active state.

This research supports the hypothesis that abnormal microglia activation plays a role in fetal alcohol spectrum disorders, however more research is needed to further understand the relationship.

Boschen, K., Ruggiero, M.J., Klintsova, A.Y., (2016) Neonatal binge alcohol exposure increases microglial activation in the developing rat hippocampus. Neuroscience 324: 355–366. DOI: 10.1016/j.neuroscience.2016.03.033

 

Stereological Study Reveals Neuron and Glia Proliferation in Hippocampus of Lithium-Treated Mice

Dentate gyruspilot

The optical fractionator probe was used to quantify the number of neurons and glia in the dentate gyrus

Doctors have used lithium to treat patients with bipolar disorder since the 1970s. Known for its efficacy in stabilizing patients’ moods by regulating manic episodes, lithium is also associated with a decreased risk of suicide. But while this naturally occurring element is the most widely prescribed medication for those suffering from bipolar disorder, scientists still have much to learn about how lithium physically affects the brain.

A recent study published in the journal Bipolar Disorders adds to the growing body of evidence that says lithium contributes to cell proliferation in parts of the brain. Conducted by scientists at the University of Mississippi and the VU University Medical Center in Amsterdam, the study revealed an increased number of neurons and glia, and increased astrocyte density in the dentate gyrus of lithium-treated mice versus controls treated with a placebo.

Using the optical fractionator probe in Stereo Investigator, the researchers quantified the number of Nissl stained neurons and glial cells, and calculated astrocyte density. The results showed twenty-five percent more neurons and twenty-one percent more glia in the denate gyrus of lithium-treated mice. They also performed a stereological examination of another brain region – the medial prefrontal cortex (mPFC), but did not witness significant differences between lithium-treated and control mice in this area.

“In this study, particular cortical regions, ie. the fascia dentata in the hippocampus and the mPFC in the cerebral cortex needed to be selected in histological sections of the mice brains,” explained Dr. Harry B.M. Uylings, “therefore the stereological counting procedure applied was the best one. Stereo Investigator greatly assisted in the counting of cells, and the software’s excel data-output was especially beneficial.”

According to the paper, the findings present a more detailed picture of lithium-induced alterations in the dentate gyrus cellular phenotype than previously available, and provide the first evidence for lithium-induced increases in glia and astrocytes.

The authors also explain that while cell number increased in the dentate gyrus of lithium-treated mice, the region’s overall volume as well as that of the greater hippocampus was unaffected by the element. The volume of the dentate gyrus and the hippocampus as a whole was measured with the Cavalieri method in Stereo Investigator.  The researchers describe the dissociation between cell proliferation and volume as “an interesting observation that warrants further investigation.”

Rajkowska, G., Clarke, G., Mahajan, G., Licht, C.M., van de Werd, H.J., Yuan, P., Stockmeier, C.A., Maji, H.K., Uylings, H.B., Differential effect of lithium on cell number in the hippocampus and prefrontal cortex in adult mice: a stereological study. Bipolar Disord. 2016 Feb;18(1):41-51. doi: 10.1111/bdi.12364.

Scientists Observe Differences Between Brains of Stressed and Unstressed Rats After Fear Conditioning

This figure illustrates the separate and combined effects of acute stress and fear conditioning/extinction on dendritic morphology of pyramidal neurons in the infralimbic region of medial prefrontal cortex. Each neuron shown is a composite made up of apical (blue) and basilar (orange) arbor near the mean of the group. The apical and basilar arbors of each composite are from different neurons. Image courtesy of Cara Wellman, PhD.

This figure illustrates the separate and combined effects of acute stress and fear conditioning/extinction on dendritic morphology of pyramidal neurons in the infralimbic region of medial prefrontal cortex. Each neuron shown is a composite made up of apical (blue) and basilar (orange) arbor near the mean of the group. The apical and basilar arbors of each composite are from different neurons. Image courtesy of Cara Wellman, PhD.

A soldier jumps at the sound of fireworks. Though there is no threat to his or her life, the blasts mimic the ones heard on the battlefield, and that fear response is not easy to forget. The process of shedding a fear response like this one is called fear extinction. Scientists think patients suffering from stress-sensitive psychopathologies, like Post-Traumatic Stress Disorder, aren’t able to suppress certain fear responses because of deficits in their brain circuitry induced by stress.

A recent study by researchers at Indiana University and the University of Haifa, in Israel, describes significant differences between the brains of stressed rats and unstressed rats.

Using Neurolucida to analyze neurons in the infralimbic cortex (IL) – a region of the brain associated with fear extinction – the research team found that stressed rats had shorter dendrites and less dendritic branching in pyramidal neurons of the IL. They also found that while stress had no affect on spine density, rats that underwent fear conditioning and extinction had decreased spine density on apical terminal branches, providing evidence that dendritic morphology in this region is sensitive to stress, while spine density may be a reflection of learning.

“Having helped colleagues set up procedures for neuron reconstructions and spine counts in labs that aren’t equipped with Neurolucida, I can tell you with complete confidence that my lab wouldn’t be nearly as productive without our Neurolucida system,” said Dr. Cara Wellman. “It makes mapping out regions of interest, identifying neurons for reconstruction, and reconstructions, and data analysis a simple and streamlined process. My students and I especially appreciate the Lucivid, which allows us to trace neurons while looking through the oculars  so much easier and clearer in my opinion than on a video monitor.”

To achieve their results, the researchers subjected rats to fear conditioning, where they learned to associate a certain tone with a footshock. Some of the rats were then exposed to an elevated platform in a brightly lit room for 30 minutes (stressed) while others returned to their home cages (unstressed). Next came extinction sessions. In a test to see if they would be able to shed the fear response associated with the stimulus, rats were placed in a space where they heard a tone but did not experience a footshock. The scientists observed that stressed rats exhibited freezing during the extinction sessions at a much higher rate than unstressed rats, leading them to believe that rats exposed to acute stress were resistant to fear extinction.

Further quantification of apical and basilar dendritic branching in the pyramidal neurons of the IL, measured with three-dimensional Sholl analysis, confirmed differences between the stressed and unstressed rats’ brains that correlated with fear behavior.

“The main findings of the current study were that acute stress, concurrent with producing resistance to extinction, produced changes in morphology of pyramidal neurons in IL,” the authors say in their paper. “These findings provide evidence that alterations in IL pyramidal neuron morphology occur quickly and differentially in response to acute stress and fear conditioning/extinction.”

Moench KM, Maroun M, Kavushansky A, Wellman C. Alterations in neuronal morphology in infralimbic cortex predict resistance to fear extinction following acute stress. Neurobiology of Stress. 3: 23-33. doi:10.1016/j.ynstr.2015.12.002

Iron Deficiency Worsens Fetal Alcohol Spectrum Disorders

An immunostained image of myelin basic protein in the cerebella of a mouse brain with an iron-sufficient diet compared with the brain of a mouse exposed to alcohol and fed an iron-insufficient diet. It shows the reduced cerebellar size due to the ID-alcohol combination. Green is MBP immunostain, blue is DAPI for nuclei.

An immunostained image of myelin basic protein in the cerebella of a mouse brain with an iron-sufficient diet compared with the brain of a mouse exposed to alcohol and fed an iron-insufficient diet. It shows the reduced cerebellar size due to the ID-alcohol combination. Green is MBP immunostain, blue is DAPI for nuclei. Image courtesy of Susan Smith, PhD.

If a pregnant woman drinks alcohol, she risks giving birth to a baby with physical and cognitive deficits – characteristics of fetal alcohol spectrum disorders. In a new study, researchers say that when the mother is low in iron, the consequences are even worse.

The scientists examined two groups of pregnant rats – one group was fed an iron sufficient diet while the other was fed a diet with insufficient iron levels. The offspring from both groups were exposed to alcohol from 4 to 9 days after birth – a time when their brains are going through a growth spurt and are particularly sensitive to alcohol. They were compared to offspring who received an iron-sufficient diet but were not exposed to alcohol. This growth spurt correlates to a growth spurt in humans that occurs during the third trimester of pregnancy.

The researchers used delay and trace eye blink classical conditioning methods to assess the offspring’s learning and memory. Learning impairments were reported in both alcohol-exposed groups regardless of their iron status, but more extreme impairments were seen in iron deficient rats compared to iron sufficient rats. After the behavioral tests were completed, the researchers studied the cerebellum and hippocampus – brain regions involved in learning and memory – at a cellular level.

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Genetic Mutation Accelerates CTE Pathology

Phosphorylated tau pS422 immunoreactive profiles in the cortex of P301Smice after repetitive mild TBI. Image courtesy of Dr. Leyan Xu.

Phosphorylated tau pS422 immunoreactive profiles (dark brown) in the cortex of P301S mice after repetitive mild TBI. Image courtesy of Dr. Leyan Xu, Department of Pathology, Johns Hopkins University.

Over the course of a football game or a boxing match, athletes may experience a series of mild concussions. Some of these athletes develop a condition known as chronic traumatic encephalopathy (CTE), a neurodegenerative disease characterized by the build-up of abnormal tau protein that eventually leads to dementia. But not every athlete develops CTE after repetitive mild traumatic brain injury, and scientists think genetic factors are involved.

In a recent study, researchers at the Johns Hopkins University School of Medicine found that the density of abnormal tau protein increased exponentially in mice that had a genetic mutation thought to cause neurodegenerative diseases. Their findings contrast with previous studies of mice without genetic mutation, where abnormal tau protein build-up did not occur. This evidence leads the scientists to infer that genetic factors play a role in the onset of CTE.

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Dying neurons in Alzheimer’s patients show signs of improvement after gene therapy

nucleus basalis of Meynert

Micrograph of cholinergic neurons in the nucleus basalis of Meynert. Image from Wikipedia.

 

Cholinergic neurons degenerate at devastating rates in Alzheimer’s disease, but Dr. Mark Tuszynski and his team at the University of California, San Diego may have found a way to slow the decline.

Their study, published in JAMA Neurology, reports that nerve growth factor gene therapy increased the size, axonal sprouting, and signaling of cholinergic neurons in 10 Alzheimer’s disease patients.

The patients were enrolled in a clinical trial between 2001 and 2012. Ex vivo and in vivo methods of gene therapy were used to deliver nerve growth factor – a protein that protects neurons and stimulates growth – to the patients. Eight received an implant of their own skin cells that were genetically modified to express nerve growth factor (ex vivo ) and two patients received injections that induced neurons already in the brain to express nerve growth factor (in vivo). In all 10 patients, gene therapy was delivered to the nucleus basalis of Meynert – part of the basal forebrain rich in cholinergic neurons that undergoes degeneration during Alzheimer’s disease. 

The patients’ survival time ranged from one to 10 years. After they had died, researchers analyzed the effects of nerve growth factor on cholinergic neurons.

The axons of cholinergic neurons, labeled with p75, grew toward the source of the nerve growth factor in all 10 patients. To determine if there was a change in cell size, researchers used the nucleator probe in Stereo Investigator to analyze cholinergic neurons of 3 patients who received gene therapy via the ex vivo method in one hemisphere – the other hemisphere was used as a control. Results from Stereo Investigator showed that cell bodies were larger in the treated hemisphere vs. the untreated hemisphere.

Finally, to find out if nerve growth factor induced signaling within cells, the researchers analyzed the amount of CREB and c-fos – markers for cell activation – in 2 patients who received nerve growth factor in vivo. An elevated amount of CREB and c-fos was found when compared to control regions. Neurons exhibiting tau pathology also expressed nerve growth factor, indicating that degenerating cells could respond to nerve growth factor gene therapy.

A phase 2 clinical study is currently under way to report cognitive outcomes in patients with Alzheimer’s disease.

“Collectively, these anatomical findings support the rationale for clinical trials to test the hypothesis that sustained growth factor delivery over time can reduce cell degeneration and stimulate cell function in chronic neurodegenerative disorders, thereby slowing functional decline,” Tuszynski, et al.

Tuszynski, M.H., Yang, J.H., Pay, M.M., Masliah, E., Barba, D., U, H.S., Conner, J.M., Kobalka, P., Roy, S., and Nagahara A.H. (2015). Nerve Growth Factor Gene Therapy: Activation of Neuronal Responses in Alzheimer Disease. JAMA Neurology, published online August 24, 2015. DOI: 10.1001/jamaneurol.2015.1807.

Munich Researchers Use WormLab to Study Blast Effects on C. elegans

 

Explosions can tear apart buildings, send shrapnel flying, and hurtle humans into the air. But explosions also cause damage in ways that aren’t as visually apparent. Scientists say the force of a blast can cause brain damage, but questions linger about how the symptoms that emerge after a blast-induced traumatic brain injury are connected to the initial trauma.

In their quest to learn more about how symptoms emerge after a traumatic blast, researchers at the Ludwig-Maximilians University of Munich, in Munich, Germany have developed an animal model of blast-related mild traumatic brain injury (br-mTBI) using C. elegans – a popular model organism alternative to vertebrate animals.

In their study, published in Frontiers in Behavioral Neuroscience, the research team used WormLab to analyze thousands of worms. They found that shockwaves either slowed the worms’ movements or rendered them paralyzed. Symptoms played out in a dose-dependent manner, meaning that worms exposed to a higher number of shockwaves displayed a higher severity of symptoms.

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Researchers Explore Spatial Memory with Stereo Investigator

The researchers quantified c-Fos positive cells in the CA1 region of the hippocampus. Image provided by Dr. Matthew Holahan.

The researchers quantified c-Fos positive cells in the CA1 region of the hippocampus. Image provided by Dr. Matthew Holahan.

Spacial memories help us navigate places like the office, the local coffee shop, or the supermarket. The hippocampus plays a key role in processing and recalling spacial memory, but as time passes, there is evidence that the anterior cingulate cortex (ACC) becomes more involved in recalling these memories. A recent paper published in PLOS ONE further investigates the ACC and found that taxing the hippocampus with spacial memory tasks accelerates the recruitment of the ACC for spacial memory recall.

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