Cracking early construction steps of the blood brain barrier

Figure 1. Early demonstration of blood-brain barrier phenomenon in developing brain.

In physiology, we often associate the terms “central” and “periphery” to refer to the brain vs the rest of the organism. This is not an anodyne dichotomy, as early 19th century injections of a dye in mice bloodstream highlighted its spreading everywhere within the organism, except in the brain (Fig. 1). In fact, a structure conveniently named the blood-brain-barrier surrounds the brain, and has two functions: protect from peripheral pathogens or toxins present in the blood, and allow nutrients to cross over to provide energy to neurons and glial cells (Fig.2).

 

Neurodegenerative diseases, ischemic strokes or other diseases such as multiple sclerosis often occur with a disruption of the blood-brain-barrier. Understanding its formation is important to investigate a cure for these disorders. In their current paper, Cottarelli and colleagues focused on the genetic determinants involved in the maturation and function of the blood-brain-barrier.

Figure 2. Blood brain barrier anatomy. From Anatomy and Physiology of the Blood–Brain Barriers, J. Abbott

The formation of a complex multicellular structure from stem cells requires the regulation of cells proliferation, migration and differentiation. These processes rely on a few key molecular signaling pathways (Fgf, hedgehog, wnt, TGFbeta, Notch). Wnt/β-catenin is one of the highly evolutionarily conserved molecular pathways that allows a cell to send information from its nucleus to cell surface receptors. Mutations in this pathway lead to abnormal development or cancer. While we know that this signaling pathway is involved in the establishment of the blood-brain-barrier, the detailed molecular mechanisms were still to elucidate. Dr Cottarelli’s work identifies a new partner of Wnt/β-catenin pathway necessary for the blood-brain-barrier development: the protein Fgfbp1 secreted by the endothelial cells of the brain and released in the basement membrane during the first weeks of age in mice. Collagen is a well known- component of conjunctive tissues. Using fluorescent microscopy techniques, Dr Cottarelli nicely highlighted a complex molecular pathway where the blood-brain-barrier maturation is enabled through collagen deposition in the vascular basement membrane. She shows that removal of Fgfbp1 gene in the blood vessels leads to a decreased signaling in the Wnt/β-catenin pathway, abnormal vascularization, delays in the establishment of the blood-brain-barrier, and abnormal cell interactions at the level of the neurovascular units. The paper also identifies a molecular mechanism linking Fgfbp1 and collagen IV in the basement membrane through the regulation of the gene Plvap (Fig 3).

 

Figure 3. Proposed model for the role of Fgfbp1 in BBB maturation.

Future studies will investigate how Fgfbp1 is involved in complex neurovascular diseases.

Azzurra Cottarelli is a postdoc in Dr Agalliu’s lab in the department of neurology. Her new paper in Development highlights her expertise in the formation of the blood-brain-barrier.

 

Mating induces transgenerational silencing in worms

Just imagine if apart from the looks one could also inherit their parents’ skills, memories, knowledge, and ideas. Sounds amazing right? However, passing down such characteristics would require transgenerational epigenetic inheritance.  The literal meaning of epigenetics is “above” or “on top of genetics”, i.e., the external modifications of the cell without any change in its DNA sequence that could turn a gene on or off and the transmission of the epigenetic marks from parents to the child is called transgenerational epigenetic inheritance.  One’s lifestyle factors, for example, diet, smoking, physical activity, alcohol consumption or even night shift work could be major contributors to the epigenetic modifications. Although the occurrence of epigenetic inheritance in humans is still a controversial debate, but it has been observed in plants, worms, mice and flies. The recent preprint by Dr. Sindhuja Devanapally and colleagues focuses on transgenerational epigenetic inheritance (TEI) and silencing in worms by reporting features that provide barrier against TEI.

Caenorhabditis elegans (C. elegans) is a transparent, small (1 mm) worm that lives in temperate soil environments with a rapid life cycle (3 days) and can be easily grown in a petri-dish while munching on bacteria as their food source. Most of these worms are hermaphrodite (with both male and female sex organs) while a few are males. These worms may look alike to the naked eyes but they differ from each other in developmental timing, lifespan and, also behavior which could be epigenetically inherited as opposed to being hard-wired in their genomes. For instance, some Pseudomonas bacteria strains are toxic food for the worms. Yet, mom-worm unlucky enough to eat the poisonous bacteria can “teach” their new born kids not to make the same mistake, thus epigenetically transferring the pathogen avoidance experience to the progeny.

RNA interference (RNAi) by double-stranded RNA (dsRNA) is a technique where RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules and has been shown to contribute to transgenerational epigenetic inheritance. The Jose lab has previously shown that dsRNA expressed within neurons of worms could enter the germline and cause transgenerational silencing. However, some worm descendants maintain the epigenetic gene silencing inherited from their ancestors for the long-term, while others lose silencing quickly. Therefore, the mechanism that can perpetuate silencing versus that can reverse it are both not clear.

RNAi

Figure 1: Transgenerational silencing of a gene is observed in descendants (no green GFP expression) for up to several generations when parents (green GFP expression) but not the kids were fed with RNAi (RNA interference, where RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules). Illustration created with BioRender.comIn this preprint, the authors fed the parent worms with double-stranded RNA (dsRNA) targeting a green fluorescent protein (GFP)-encoding gene expressed in the worm germline and monitored the maintenance of gene silencing in their unfed descendants  (Fig. 1). While this GFP expression was turned off in initial generations, it almost always came back in the later generations except in one peculiar case. The authors  discovered that RNAi against GFP when expressed as part of a rare recombinant two gene operon, named T (containing GFP and mCherry fluorescent proteins), showed permanent RNA-based silencing. They reported that such silencing can also be triggered without using dsRNA and simply by mating dad-worms expressing T with mom-worms (hermaphrodites) lacking T expression. Because this kind of inducible permanent silencing was never reported previously, the authors introduced this phenomenon as mating-induced silencing. Mating induced silencing of T could be maintained for more than 300 generations without selection beyond second generation, thus making it the first ever study to report persistent silencing without external triggers. As the authors report, this contrasts dramatically with the genes expressed in the germline that can be silenced for a few generations by RNAi or trans effects of mating-induced silencing. Follow up experiments confirmed that maternal T can provide a protective signal that prevents paternal T silencing, suggesting that the germline has evolved to prevent permanent silencing potentially to prevent negative responses to temporary change in the environment.

According to the germline immortality concept, unlike somatic cells, the germline cells are well protected from the environment and can be passed on indefinitely across generations. However, Devanapally and colleagues in the current study reported that the expression of genes (not all but rare examples like T) within the germline can potentially be changed for hundreds of generations without any external triggers. This highlights how worms have adopted fascinating epigenetic mechanisms to accelerate evolution yet keeping the DNA sequence unchanged. Yet, the sheer infrequency at which permanent changes occur shows how impenetrable the germline is to permanent changes and the germline’s capacity to revert back to ancestral gene expression states. Thus, this study points to an organism’s ability to preserve persistence of gene expression, resulting in the preservation of the species.

Whether such rare examples of transgenerational epigenetic inheritance also occur in mammals, especially humans, is still up for debate. Epigenetic modifications have to occur in sperm or egg cells in order to pass to the next generation. Yet, most of these modifications in sperm and eggs get erased upon fertilization, resetting it to default and thus the next generation starts from scratch and makes its own epigenetic modifications. However, it is believed that some of these epigenetic modifications can escape this erasure and are passed on to the progeny. A study published in Nature Journal in 2013 reported that the mice-parents exposed to smell-fear conditioning (smell followed by electric shock) could pass their trauma to the next generation. Although rare, this opens up the possibility that indeed parents could pass on their experience, skills or even fear to the next generation. It will be fascinating to identify the mechanisms by which environmental information is transgenerationally inherited in humans.

Dr. Sindhuja Devanapally is a Postdoctoral Research Scientist in the Department of Biochemistry and Molecular Biophysics, and co-chair of the Networking and Community Building committee of CUPS.

 

 

Plasticity inception in a nutshell

Have you ever realized that you remember experiences associated with strong emotions more vividly? For example, you probably remember what you ate at your (or a close friend’s) wedding, but not last Tuesday. However, these persistent memories are not always pleasant. People exposed to actual or threatened death, serious injury, or sexual violence can develop Post-Traumatic Stress Disorder (PTSD), which involves recurring memories or dreams of the traumatic event, bodily reactions to reminders and active avoidance of those reminders. Treatment for PTSD combines psychotherapy and medication, and it aims at enabling the person to understand their trauma and detach the triggers from the responses.

The area in your brain responsible for the formation of such emotional memories is called the amygdala (from the Greek word for almond, due to its shape, Fig. 1). It can modify the way it will respond to similar stimuli in the future, and it can also affect how other brain areas, like the medial prefrontal cortex or the hippocampus, do as well. This ability to change and adapt is called plasticity, and it can start with something as “simple” as a synaptic connection becoming stronger or weaker. There are higher levels of plasticity, though. If changes alter the potential response of a region to a future challenge, this plasticity of plasticity is called metaplasticity.

Human and rodent brain with highlighted amygdala, medial prefrontal cortex and hippocampus.
Fig. 1. Depiction of a human and a rodent brain. Highlighted areas are responsible for establishing emotional memories, fear conditioning and extinction. Modified from Sokolowski and Corbin 2012.

In the recent review “Intra-Amygdala Metaplasticity Modulation of Fear Extinction Learning”, CUIMC postdoc Dr. Rinki Saha and colleagues provide a comprehensive account of recent literature on metaplasticity in the amygdala in the context of fear conditioning, and how it may lead to plasticity in other connected brain regions.

Fear conditioning is a classic rodent model in neuroscience research that allows scientists to study the mechanisms that lead to associations between neutral stimuli and unpleasant stimuli. The general experimental layout is as follows: first, a neutral stimulus (a light or a tone, for example) is consistently paired to precede an aversive stimulus (like an electric foot shock). After this exposure, animals learn that the neutral stimulus (called conditioned stimulus) predicts the aversive one (called unconditioned stimulus) and they develop a fear response which they perform right after the neutral stimulus (life freezing in place). The experiment can continue to study how they learn to dissociate them once the stimuli stop being paired. For this second part, called fear extinction learning, the neutral stimulus is presented by itself (without pairing it to the aversive one), and researchers measure the time it takes the animal to stop performing the fear response.

In order to study the amygdala’s role in fear extinction, scientists can inject different drugs into it with very fine syringes (in a procedure called stereotaxic surgery, Fig. 2). By either activating or inhibiting different signaling pathways, they can elucidate what roles those molecules play in the fear extinction process. In addition, experiences like stress and trauma can interfere with this extinction learning, as evidenced in people who suffer from PTSD and in rodent models exposed to different stressful situations, both acute and chronic.

Depiction of a stereotaxic surgery in a rodent. Detail of injection in the amygdala.
Fig. 2. Depiction of a stereotaxic surgery in a rodent. The anesthetized animal is fixed on the frame of the stereotaxic instrument, which has very accurate rulers for the three dimensions. A very fine syringe is introduced through the skull into the brain to administer the drug or virus in a very precise way.
Made with BioRender.

This paradigm has been used by many to study metaplasticity, where the change that occurs is not a modification of the baseline response but rather of the response to a subsequent plasticity-inducing stimulation. For example, Dr. Saha herself showed that it is possible to alter fear extinction learning by injecting a virus into a subregion of the amygdala that disrupts inhibitory synapses. Importantly, this happened without modifying the initial fear conditioning or the anxiety level of the animals. In addition, they also showed that those alterations in inhibitory synapses in the amygdala led to independent changes in the medial prefrontal cortex, hindering its intrinsic plasticity. The same intervention caused increased resilience to acute trauma and improved the performance of a task dependent on another brain region, the hippocampus. Hence, a very targeted intervention in the amygdala can cause an array of effects across multiple brain areas.

This body of research has tremendous implications in our understanding of the brain and how to treat its diseases. On a very pragmatic sense, it should serve as a cautionary tale for researchers to take into account and consider the potential for “undesired” plasticity in more than one place as a response to certain interventions. But more importantly, it opens up potential therapeutic strategies for trauma-related disorders like PTSD, stress or fear. Changes in one small region can lead to widespread effects through its connections to other brain areas. Hopefully, we are a little bit closer to tricking the brain into equating those traumatic memories with what you ate last Tuesday.

 

Dr. Rinki Saha is a Postdoctoral Research Fellow in the Department of Psychiatry researching  stress, and one of CUPS’ social media managers.