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New evidence has shown how coronavirus has caused much damage to the brain. There is a new evidence that shows that COVID-19 assault on the brain I has the power to be multipronged. What this means is that it can attack on certain Brain cells such as reduce the amount of blood flow that the brain needs to the brain tissue.
Along with brain damage COVID-19 has also caused strokes and memory loss. A neurologist at yell University Serena Spudich says, “Can we intervene early to address these abnormalities so that people don’t have long-term problems?”
We’re on 80% of the people who have been hospitalized due to COVID-19 have showed brain symptoms which seem to be correlated to coronavirus.
At the start of the pandemic a group of researchers speculated that coronavirus they can damage the brain by infecting the neurons in the cells which are important in the process of transmitting information. After further studies they found out that coronavirus has a harder time getting past the brains defense system and the brain barrier and that it does not affect the neurons in anyway.
An expert in this study indicated that a way in which SARS-CoV-2 may be able to get to the brain is by going through the olfactory mucosa which is the lining of the nasal cavity. It is found that this virus can be found in the nasal cavity which is why we swab the nose one getting tested for COVID-19.
Spudich quotes, “there’s not a tonne of virus in the brain”.
Recent studies indicate that SARS-CoV-2 have ability to infect astrocytes which is a type of cell found in the brain. Astrocytes do quite a lot that supports normal brain function,” including providing nutrients to neurons to keep them working, says Arnold Kriegstein, a neurologist at the University of California, San Francisco.
Astrocytes are star-shaped cells in the central nervous system that perform many functions, including providing nutrients to neurons.
Kriegstein and his fellow colleagues have found that SARS-CoV-2 I mostly infects the astrocytes over any of the other brain cells present. In this research they expose brain organoids which is a miniature brain that are grown from stem cells into the virus.
As quoted in the article” a group including Daniel Martins-de-Souza, head of proteomics at the University of Campinas in Brazil, reported6 in a February preprint that it had analysed brain samples from 26 people who died with COVID-19. In the five whose brain cells showed evidence of SARS-CoV-2 infection, 66% of the affected cells were astrocytes.”
The infected astrocytes could indicate the reasoning behind some of the neurological symptoms that come with COVID-19. Specifically, depression, brain fog and fatigue. Kreigstein quotes, “Those kinds of symptoms may not be reflective of neuronal damage but could be reflective of dysfunctions of some sort. That could be consistent with astrocyte vulnerability.”
A study that was published on June 21 they compared eight different brands of deceased people who did have COVID-19 along with 14 brains as the control. The results of this research were that they found that there was no trace of coronavirus Brain infected but they found that the gene expression was affected in some of the astrocytes.
As a result of doing all this research and the findings the researchers want to know more about this topic and how many brain cells need to be infected for there to be neurological symptoms says Ricardo Costa.
Further evidence has also been done on how SARS-CoV-2 can affect the brain by reducing its blood flow which impairs the neurons’ function which ends up killing them.
Pericytes can be found on the small blood vessels which are called capillaries and are found all throughout the body and in the brain. In a February pre-print there was a report about how SARS-CoV-2 can infect the pericyte in the brain organoids.
David Atwell, a neuroscientist at the University College London, along with his other colleagues had published a pre-print which has evidence to show that SARS-CoV-2 odes In fact pericytes behavior. I researchers saw that in the different part of the hamsters brain SARS-CoV-2 blocks the function of receptors on the pericytes which ultimately causes the capillaries found inside the tissues to constrict.
As stated in the article, It’s a “really cool” study, says Spudich. “It could be something that is determining some of the permanent injury we see — some of these small- vessel strokes.”
Attwell brought to the attention that the drugs that are used to treat high blood pressure may in fact be used in some cases of COVID-19. Currently there are two clinical trials that are being done to further investigate this idea.
There is further evidence showing that the neurological symptoms and damage could in fact be happening because of the bodies on immune system reacting or misfiring after having COVID-19.
Over the past 15 years it has become evident that people’s immune system’s make auto antibodies which attack their own tissues says Harald Prüss in the article who has a Neuroimmunologist at the German Center for neurogenerative Diseases in Berlin. This may cause neuromyelitis optica which is when you can experience loss of vision or weakness in limbs. Harald Prüss summarized that the autoantibodies can pass through the blood brain barrier and ultimately impact neurological disorders such as psychosis.
Prüss and his colleagues published a study last year that focused on them isolating antibodies against SARS-CoV-2 from people. They found that one was able to protect hamsters from lung damage and other infections. The purpose of this was to come up with and create new treatments. During this research they found that some of the antibodies from people. They found that one was able to protect hamsters from lung damage and other infections. The purpose of this was to come up with and create new treatments. During this research they found that some of the antibodies can bind to the brain tissue which can ultimately damage it. Prüss states, “We’re currently trying to prove that clinically and experimentally,” says Prüss.
Was published online in December including Prüss sorry the blood and cerebrospinal fluid of 11 people who were extremely sick with COVID-19. These 11 people had neurological symptoms as well. All these people were able to produce auto antibodies which combined to neurons. There is evidence that when the patients were given intravenous immunoglobin which is a type of antibody it was successful.
Astrocytes, pericytes and autoantibodies we’re not the only pathways. However it is likely that people with COVID-19 experience article symptoms for many reasons. As stated, In the article, Prüss says a key question is what proportion of cases is caused by each of the pathways. “That will determine treatment,” he says.
Comparing COVID-19 Vaccine Schedule Combinations, or “Com-COV” – First-of-its-Kind Study will explore the Impact of using eight different Combinations of Doses and Dosing Intervals for Different COVID-19 Vaccines
Compared to other mammals, humans have the largest cerebral cortex. A sheet of brain cells that folds in on itself multiple times in order to fit inside the skull, the cortex is the seat of higher functions. It is what enables us to process everything we see and hear and think.
The expansion of the cerebral cortex sets humans apart from the rest of their fellow primates. Yet scientists have long wondered what mechanisms are responsible for this evolutionary development.
New research from the Kosik Molecular and Cellular Neurobiology Lab at UC Santa Barbara has pinpointed a specific long nocoding ribonucleic acid (lncRNA) that regulates neural development (ND). The findings appear in the journal Neuron.
“This lncND, as we’ve called it, can be found only in the branch of primates that leads to humans. It is a stretch of nucleotides that does not code a protein,” said senior author Kenneth S. Kosik, the Harriman Professor of Neuroscience Research in UCSB’s Department of Molecular, Cellular, and Developmental Biology. “We demonstrate that lncND is turned on during development and turned off when the cell matures.”
Lead author Neha Rani, a postdoctoral scholar in the Kosik Lab, idenfitied several binding sites on lncND for another type of RNA called a microRNA. One of them, called microRNA-143, binds to lncND.
“We found that lncND could sequester this microRNA and in doing so regulate the expression of Notch proteins,” Rani said. “Notch proteins are very important regulators during neuronal development. They are involved in cell differentiation and cell fate and are critical in the neural development pathway.”
Kosik describes lncND as a platform that binds these microRNAs like a sponge. “This allows Notch to do what it’s supposed to do during development,” he explained. “Then as the brain matures, levels of lncND go down and when they do, those microRNAs come flying off the platform and glom onto Notch to bring its levels down. You want Notch levels to be high while the brain is developing but not once maturation occurs. This lncND is an elegant way to change Notch levels quickly.”
To replicate these cell culture results, Rani used human stem cells to grow neurons into what is called a mini brain. In this pea-sized gob of brain tissue, she identified a subpopulation — radial glial cells (neuronal stem cells) and other neural progenitors — responsible for making lncND.
But the researchers wanted to see the radial glial cells in actual human brain tissue, so they turned to colleagues in the Developmental & Stem Cell Biology Graduate Program at the UC San Francisco School of Medicine. Using in situ hybridization, UCSF scientists found lncND in neural precursor cells but not in mature neurons.
“It was right where we thought it would be in brain tissue,” said Kosik, who is also the co-director of UCSB’s Neuroscience Research Institute. “But we still had one more thing we had to do because people would still not be satisfied that we had done everything possible to show that lncND was really doing something functionally.”
So the UCSF team introduced lncND into the fetal brain of a gestating mouse. Green fluorescent protein labeling allowed them to see the early development pattern and show that lncND, which ordinarily is not present in mice — lncND is present only in some primates including humans — had a functional effect on development.
“When we overexpressed lncND in the mouse fetus, we actually affected development in the predicted manner,” Kosik said. “The early developmental pattern was shifted toward more precursor cells, even though the mouse does not make lncND at all.”
According to Kosik, this work not only identifies a very critical gene for human brain development but also offers a clue about a component that likely contributed to brain expansion in humans. “We have shown that lncND might be an important player in human brain expansion, which is exciting in itself,” Rani said. “Another interesting aspect of this work is that lncND appears to help regulate the key developmental pathway of Notch signaling.”
Figure 1: Cells in which NeuroD1 is turned on are reprogrammed to become neurons. Cell nuclei are shown in blue (Höchst stain) and neurons are shown in red (stained with neuronal marker TUJ1). [A. Pataskar,J. Jung, V. Tiwari]
Researchers at the Institute of Molecular Biology (IMB) in Mainz, Germany say they have unraveled a complex regulatory mechanism that explains how a single gene can drive the formation of brain cells. Their study (“NeuroD1 reprograms chromatin and transcription factor landscapes to induce the neuronal program”), published in The EMBO Journal, is an important step toward a better understanding of how the brain develops. It also harbors potential for regenerative medicine, according to the scientists.
Neurodegenerative disorders, such as Parkinson’s disease, are often characterized by an irreversible loss neurons. Unlike many other cell types in the body, neurons are generally not able to regenerate by themselves, so if the brain is damaged, it stays damaged. One hope of developing treatments for this kind of damage is to understand how the brain develops in the first place, and then try to imitate the process. However, the brain is also one of the most complex organs in the body, and very little is understood about the molecular pathways that guide its development.
Figure 2: Diagram showing how NeuroD1 influences the development of neurons. During brain development, expression of NeuroD1 marks the onset of neurogenesis. NeuroD1 accomplishes this via epigenetic reprogramming: neuronal genes are switched on, and the cells develop into neurons. TF: transcription factor; V: ventricle; P: pial surface. [A. Pataskar, J. Jung, V. Tiwari]
ijay Tiwari, Ph.D, and his group have been investigating a central gene in brain development, NeuroD1. This gene is expressed in the developing brain and marks the onset of neurogenesis.
In their research article, Dr. Tiwari and his colleagues have shown that during brain development NeuroD1 is not only expressed in brain stem cells but acts as a master regulator of a large number of genes that cause these cells to develop into neurons. They used a combination of neurobiology, epigenetics, and computational biology approaches to show that these genes are normally turned off in development, but NeuroD1 activity changes their epigenetic state in order to turn them on. Strikingly, the researchers show that these genes remain switched on even after NeuroD1 is later switched off. They further show that this is because NeuroD1 activity leaves permanent epigenetic marks on these genes that keep them turned on, in other words it creates an epigenetic memory of neuronal differentiation in the cell.
“Our research has shown how a single factor, NeuroD1, has the capacity to change the epigenetic landscape of the cell, resulting in a gene expression program that directs the generation of neurons,” wrote the screenplay investigators.
“This is a significant step toward understanding the relationship between DNA sequence, epigenetic changes and cell fate. It not only sheds new light on the formation of the brain during embryonic development but also opens up novel avenues for regenerative therapy,” says Dr. Tiwari.
NEUROD1 neuronal differentiation 1 [ Homo sapiens (human) ]
Summary This gene encodes a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors. The protein forms heterodimers with other bHLH proteins and activates transcription of genes that contain a specific DNA sequence known as the E-box. It regulates expression of the insulin gene, and mutations in this gene result in type II diabetes mellitus. [provided by RefSeq, Jul 2008]
Neurogenic differentiation 1 (NeuroD1), also called β2,[1] is a transcription factor of the NeuroD-type. It is encoded by the human gene NEUROD1.
It is a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors. The protein forms heterodimers with other bHLH proteins and activates transcription of genes that contain a specific DNA sequence known as the E-box. It regulates expression of the insulin gene, and mutations in this gene result in type II diabetes mellitus.[2]
After their generation and specification in periventricular regions, neuronal precursors maintain an immature and migratory state until their arrival in the respective target structures. Only here are terminal differentiation and synaptic integration induced. Although the molecular control of neuronal specification has started to be elucidated, little is known about the factors that control the latest maturation steps. We aimed at identifying factors that induce terminal differentiation during postnatal and adult neurogenesis, thereby focusing on the generation of periglomerular interneurons in the olfactory bulb. We isolated neuronal precursors and mature neurons from the periglomerular neuron lineage and analyzed their gene expression by microarray. We found that expression of the bHLH transcription factor NeuroD1 strikingly coincides with terminal differentiation. Using brain electroporation, we show that overexpression of NeuroD1 in the periventricular region in vivo leads to the rapid appearance of cells with morphological and molecular characteristics of mature neurons in the subventricular zone and rostral migratory stream. Conversely, shRNA-induced knockdown of NeuroD1 inhibits terminal neuronal differentiation. Thus, expression of a single transcription factor is sufficient to induce neuronal differentiation of neural progenitors in regions that normally do not show addition of new neurons. These results suggest a considerable potential of NeuroD1 for use in cell-therapeutic approaches in the nervous system.
Determination of neuronal subtypes is an early event that coincides with cell cycle exit (1, 2). However, after their generation, new neurons have to remain immature for prolonged periods, allowing their migration to final destinations where terminal differentiation occurs (3). Little is known about the factors that maintain the precursor state or induce terminal differentiation.
Olfactory neurogenesis is particularly suited to approach these late steps in neuronal differentiation. Here, stem cell populations first located in the ventricular zone and after the establishment of an ependymal layer positioned in subventricular zone (SVZ) generate migratory neuroblasts throughout life (4). These perform long-distance chain migration via the rostral migratory stream (RMS) into the olfactory bulb (OB), where they migrate into the granule cell layer (GCL) and the glomerular layer (GL) to differentiate into GABA- and dopaminergic neurons (4, 5). Thus, in this system, generation of neurons is permanent and the consecutive steps in the neurogenic sequence are spatially separated.
Determination of newly generated neurons has been studied intensively over the past years. For example, it has been demonstrated that defined areas surrounding the lateral ventricle contain predetermined stem cells that give rise to defined subsets of interneurons (6, 7). Several transcription factors have been implicated in the specification of the different neuronal populations. The zinc finger transcription factor sp8, for instance, appears to be involved in the generation of interneurons expressing calretinin (8), and analysis of Sall3 mutant mice (9) points to a role of this factor in the dopaminergic, tyrosine hydroxylase–positive lineage (10). Furthermore, it appears that interneuron diversity relies on the combinatorial expression of such transcription factors. This is exemplified by Pax6 and Dlx2, which have been shown to interact in the determination of adult generated neuronal precursors toward a dopaminergic fate (9, 11, 12). All of these transcriptional regulators are expressed early during the neurogenic process and remain present until terminal differentiation occurs.
We aimed at the identification of transcription factors that induce terminal differentiation of postnatal generated neurons in the OB. To do so we isolated neuronal precursors and differentiated interneurons from the periglomerular lineage of the OB and compared their gene expression by microarray. We established that the expression of NeuroD1, a bHLH transcription factor that has been implicated in neuronal differentiation in several experimental systems (13–17), coincides with the passage from neuronal precursor to mature interneurons. Functionally, we show that premature expression of NeuroD1 in vitro and in vivo induced highly efficiently the differentiation of forebrain progenitors. In vivo, this leads to the transitory appearance of ectopic neurons in the SVZ, RMS and striatum. Conversely, knockdown of NeuroD1 specifically inhibits terminal maturation of periglomerular neurons in the OB. Thus, NeuroD1 is both necessary and sufficient to induce key steps in terminal neuronal differentiation.
NeuroD1 Is Specifically Expressed in Mature GL Interneurons.
Subpopulations of neuronal precursors destined for the GCL and GL of the OB are generated by regionally defined stem cell populations in the periventricular region but migrate intermingled in the RMS to the OB. Once there, cells resegregate: granule cell precursors terminate their migration in the GCL, whereas the smaller population of periglomerular neuron precursors traverses this layer and the mitral cell layer (MCL) to invade the peripherally located GL (Fig. 1A). Thus, at a given time point, the GL contains both mature periglomerular neurons and their specific progenitors. Based on this spatial organization we isolated these two populations, concurrently depleting glial cells.
We devised a three-step strategy based on the following: (i) microdissection followed by enzymatic dissociation of the postnatal GL, (ii) depletion of contaminating glial cells by magnetic activated cell sorting (MACS) using an A2B5 specific antibody (18), (iii) separation of PSA-NCAM expressing cells (19) from the remaining fraction containing the mature neurons (Fig. 1B). The same purification strategy was applied to tissue microdissected from the P2 periventricular region (18). Characterization of the different cell population after sorting was performed via immunocytochemistry using the markers used for sorting (A2B5 and PSA-NCAM) as well as the differentiation marker Gad65 (18) (Fig. S1). Thus, as starting material we obtained highly enriched mature OB periglomerular interneurons (PGN), their immature progenitors (PGP), as well as a mixed population of generic progenitors (GP) from the SVZ/RMS.
Fig. 1.
Expression of NeuroD1 in the olfactory neurogenic system (A) DAPI-stained coronal section through the olfactory bulb of P5. (B) Strategy to isolate neuronal populations at different steps of their maturation. (C) Relative changes in gene expression for selected genes. Expression in GP was considered baseline, and changes are expressed as fold difference. (D–F) NeuroD1 in situ hybridization on sections from P5 mouse brain. No signal was detected along the lateral ventricle or in the RMS (D). In the olfactory bulb, individual NeuroD1+ cells were present in the GCL, whereas the MCL and the GL contained higher amounts (E, high magnification in F). A similar expression pattern was found after β-gal reaction on NeuroD1-lacZ-knockin tissue (G). (Scale bar: 200 μm in A; 100 μm inD and E; 20 μm in F and G).
Based on the purified and characterized cell populations, we performed microarray analyses to gain insight into the changes in gene expression during the neurogenic process. Investigation of expression dynamics of genes associated with either the precursor status or neuronal differentiation (Fig. S2 A and B) were used to validate the approach. Furthermore, these data were compared with those from an already available Serial Analysis of Gene Expression (SAGE) study (20).
Serial Analysis of Microarray (SAM) demonstrated the presence of groups of genes with comparable expression patterns (Fig. S2 C–E). Interestingly, only a relatively small fraction of genes were absent in the immature cell populations GP and PGP but highly represented in mature PGN (Fig. S2E). One of the genes showing such a pattern was NeuroD1, which was expressed more than 50-fold higher in PGN than in the immature populations (Fig. 1C). This was in agreement with the above-cited SAGE data, showing that NeuroD1 expression was below the detection level in neuronal precursors of the adult SVZ (20). Thus, expression of NeuroD1 was absent from precursors but coincided with terminal neuronal differentiation.
This late expression of NeuroD1 was in contrast to that of factors that have been functionally implicated in the specification of PGN, including Pax6, Sp8 and Sall3, which were expressed in both the immature populations and in the mature neurons (Fig. 1C; in situ hybridization for Pax6 in Fig. S3). Only Dlx2 showed a moderate increase in the PGN lineage outgoing, however, from an already considerable baseline level in migrating precursors (12) (Fig. 1C).
Next we analyzed the expression of NeuroD1 using in situ hybridization on P5 forebrain sections. Strong expression was found in the GL, whereas weaker expression was observed in the GCL and MCL (Fig. 1 Eand F). The transcript was undetectable in the periventricular region and the RMS (Fig. 1D). This staining was confirmed using NeuroD1-lacZ knockin mice (21) (Fig. 1G). In conclusion, these data demonstrated the absence of NeuroD1 from immature cells of the system and its strong expression in mature PGN. This pattern was coherent with a function in terminal neuronal differentiation.
NeuroD1 Induces Neuronal Differentiation in Vitro.
We studied the neurogenic potential of NeuroD1 in primary cultured neural stem cells using the neurosphere assay. In parallel to NeuroD1, we performed all experiments under the same conditions using the transcription factor Pax6, a well-described neurogenic signal in the system (9, 11, 12), to control for specificity of the observed effects. Neurosphere cells were coelectroporated with NeuroD1 or Pax6 expression vectors and GFP immediately before plating in differentiation conditions. One week after transfection, in the control condition, 14 ± 1% of the GFP-positive cells coexpressed the early neuronal marker Tuj1 (Fig. S4 A and D) whereas NeuroD1 induced Tuj1 expression in virtually all cells (98.0 ± 2%, Fig. S4 B and D). Pax6 gain-of-function led to an intermediate value (60.0 ± 3%, Fig. S4 C and D). NeuN, a later neuronal marker (22), was expressed by 21.1 ± 1% of the Tuj1-positive cells in the control situation (Fig. S4 E and H) but induced by NeuroD1 in almost all cells (93.9 ± 2%; Fig. S4 F and H). Surprisingly, Pax6 expression led to nearly complete disappearance of NeuN (1.7 ± 0.3%; Fig. S4 G and H). We investigated the induction of subtype specific markers by NeuroD1. Whereas tyrosine hydroxylase showed no augmentation, we found a 20% increase in calretinin labeling, in agreement with previous findings (23).
Next we investigated morphological parameters like process length as well as density and length of filopodia. Both NeuroD1 and Pax6 induced a significant, greater than 2-fold increase in process length (Fig. S4 I and L). We analyzed dendritic filopodia, structures that are believed to be precursors of dendritic spines (24). Expression of NeuroD1 induced a doubling in density and length of filopodia (Fig. S4 N, P, and Q). Interestingly, Pax6 reduced filopodia density to a level significantly below that of controls (Fig. S4 O and P), whereas length of the few remaining filopodia was not affected (7.0 ± 0.4 μm; Fig. S4Q).
Thus, the expression of NeuroD1 in neurosphere amplified neural stem cells induced neuronal commitment as well as morphological characteristics of mature neurons. Like NeuroD1, Pax6 favored neuronal commitment but appeared to actively suppress certain characteristics of terminal neuronal differentiation.
NeuroD1 Induces Ectopic Neurons in Vivo.
We asked whether NeuroD1 was also sufficient to induce neuronal differentiation in vivo. We used postnatal forebrain electroporation, an approach that allows efficient genetic manipulation of neural stem cells along the lateral ventricles and, consequently, of all transitory or permanent cell populations that are generated in the olfactory neurogenic process (25). The NeuroD1 expression vector or empty control plasmids were coelectroporated together with a GFP-containing vector that allowed visualization of transfected cells and their progeny at high resolution. Consequences of NeuroD1 gain-of-function were analyzed at 2, 4, 6, 8, and 15 days postelectroporation (dpe). As for the in vitro studies, results were compared with the effects of Pax6 gain-of-function.
At 2 dpe of a control vector into the lateral wall of the forebrain ventricle, 9.8 ± 1.3% (Fig. 2 A and K) of the GFP-expressing cells were localized in the VZ and had the morphology of radial glia (RG) (25). The majority of the GFP + cells, representing mainly neuronal precursors, were localized in the SVZ. Electroporation of a NeuroD1 expression vector induced a loss of GFP-positive RG cells (3.7 ± 0.5%; Fig. 2 B and K). The remaining cells in the VZ showed lower GFP levels than in controls (Fig. 2 A and B asterisks).
Fig. 2.
NeuroD1 induces neuronal morphology in vivo. Effect of NeuroD1 gain-of-function at different time points postelectroporation. (A and B) Coronal forebrain sections at the level of the lateral ventricle at 2 dpe. In the control condition, strongly GFP labeled RG are present in the VZ (A, asterisk). Expression of NeuroD1 induced a relative loss of radial glia and fainter GFP label (B, asterisk). (C and D) Coronal sections at the level of the lateral ventricle at 4 dpe. NeuroD1 expression induced an accumulation of transfected cells in the SVZ (D) and the almost total disappearance of radial glia (D). (E–F′) Sagittal sections of the RMS at 4 dpe. In the control situation, cells migrated toward the OB and presented the bipolar morphology specific of migrating precursors (E, E′, arrowheads). NeuroD1 electroporation induced loss of tangential orientation, induction of complex branching (F, F′, arrowhead), and invasion of the surrounding tissues (F, arrowheads). (G and H) Coronal section at the level of the olfactory bulb at 4 dpe. Although the majority of cells have reached the OB in the control situation (G), only a few cells were located in the center of the OB in the presence of NeuroD1 (H). (I and I′) Examples of cells presenting neuronal morphology in the SVZ at 4 dpe. (J) High magnification showing the presence of filopodia covering NeuroD1-expressing cells (arrowheads). (K) Quantification of GFP-positive cells presenting radial glia cell morphology along the lateral ventricle at 2 and 4 dpe. Control: 9.8 ± 1.3% (n= 6) at 2 dpe; 24 ± 11.8% at 4 dpe (n = 3); NeuroD1: 3.7 ± 0.5% at 2 dpe (n = 6); 1.6 ± 0.7% at 4 dpe (n = 3). (l) Distribution of the GFP-positive cells along the rostrocaudal axis. NeuroD1 expressing cells accumulated in proximal parts of the system. (M) Morphological analysis of cells in the SVZ/RMS. Three different classes were defined: (i) bipolar cells presenting tangential orientation, (ii) spherical cells, and (iii) branched cells presenting multiple processes in various directions (compare I). NeuroD1-expressing cells presented a highly branched morphology. Control: bipolar, 80.4%; spherical, 19.5%; branched, 0% (n = 133 cells). NeuroD1: bipolar, 5%; spherical, 16.8%; branched, 78% (n = 119 cells). Statistics: Mann-Whitney test. ns, not significant. **P < 0.01; ***P < 0.005. (Scale bar: 100 μm in E, F, G,and H; 25 μm in A, B, C, D,E, and F’; 10 μm in I; 5 μm in J.)
At 4 dpe, in the control situation, considerable amounts of strongly GFP+ RG cells were still present in the VZ (Fig. 2C asterisks), whereas NeuroD1 expression induced an almost complete loss of RG cells (Fig. 2 Dand K). At this time point, control cells were found along the entire SVZ and RMS. They showed generally tangential orientation and the typical morphology of migratory neuronal precursors. Large amounts of such cells were also found in the center of the OB (Fig. 2 G and L). NeuroD1 expression induced an accumulation of GFP-labeled cells in the SVZ (Fig. 2 D and L) at the expense of cells in the RMS (Fig. 2H,quantified in Fig. 2L). The accumulating cells did not have the appearance of migrating precursors but displayed complex multibranched morphologies (Fig. 2 F and F′, examples in Fig. 2 I and I′, quantified inFig. 2M). All principal processes of these cells were covered with small protrusions resembling filopodia (Fig. 2J). Such morphologically complex cells, strongly resembling neurons, were also predominant in and along proximal parts of the RMS (Fig. 2F′). Interestingly, considerable amounts of multibranched cells were found outside of the periventricular region and the RMS, invading neighboring structures such as the striatum (Fig. 2F, arrows). There was a clear correlation between the quantity of transgene expression, as visualized by GFP fluorescence, and the above parameters. Thus, NeuroD1 induced dose-dependently a neuron-like morphology in cells in the SVZ, RMS, and surrounding tissues.
We characterized the NeuroD1 induced neuron-like cell population in the periventricular region using neuronal and glial markers (Fig. 3; examples in Fig. S5). Doublecortin (DCX), a microtubule-associated protein expressed in migratory neuronal precursors (26), was seen in 75.2 ± 4.5% of the cells in the control situation but showed a significant increase after expression of NeuroD1 (91.7 ± 2.2%). NeuN, a marker for most mature neuronal cell types in the brain (22) was low in controls (5.2 ± 1.4%, n = 8) but strongly induced by NeuroD1 (65.9 ± 4.5%, n = 9). Map2, a later generic neuronal marker (27), was also rare in control cells (14.1 ± 1.4%, n = 3) but highly expressed in the NeuroD1 condition (61.9 ± 2.7%, n = 3). GFAP and Olig2 did not show significant alterations due to NeuroD1 expression. Thus, the NeuroD1-induced ectopic cells with neuronal morphology in the SVZ and RMS showed molecular characteristics of neurons.
Fig. 3.
NeuroD1 induces generic neuronal markers in vivo Molecular phenotype of the cells located in the periventricular region (level 4 in Fig. 2l). Quantification representing the percentage of GFP-positive cells expressing the respective markers. DCX: control, 75.2 ± 4.5%, n = 5; NeuroD1, 91.7 ± 2.2%, n = 5. NeuN: control, 5.2 ± 1.4%, n = 8; NeuroD1, 65.9 ± 4.5%, n = 9. Map2: control, 14.1 ± 1.4%, n = 3; NeuroD1, 61.9 ± 2.7%, n = 3. Olig2: control, 6.8 ± 5%, n = 3; NeuroD1, 2.5 ± 0.5%, n = 3. GFAP: control, 0%, n = 3; NeuroD1, 0%, n = 2. Errors bars indicate SEM. Statistics: DCX and Map2, unpaired ttest; NeuN, Mann-Whitney test. ns, not significant. *P < 0.05; **P < 0.01; ***P < 0.005.
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NeuroD1 Is Necessary For OB Interneuron Differentiation in Vivo.
Next we asked whether NeuroD1 is essential for the generation of PGN. Given that NeuroD1 deficiency in mice is generally associated with perinatal lethality (14, 15, 21), we used a strategy based on RNAi in concert with postnatal in vivo electroporation to knock down NeuroD1 in the olfactory bulb neurogenic system. For validation, three different NeuroD1 specific shRNA vectors were cotransfected with a NeuroD1 expression construct into COS-7 cells. Western blot analysis demonstrated that two of the shRNAs, sh775 and sh776, efficiently inhibited production of the NeuroD1 protein, whereas sh777 induced a less efficient downregulation (sh775, 94.6%; sh776, 96.9%; sh777, 78.4%; corrected for loading against αtubulin; Fig. 4A). All three shRNAs were used for further in vivo studies.
Fig. 4.
In vivo terminal neuronal differentiation of PGC is impaired in absence of NeuroD1. (A) Western blot analysis of protein extracts from cos-7 cells transfected with NeuroD1 or in combination with different NeuroD1 specific shRNAs. sh775 and sh776 strongly repressed NeuroD1 protein expression (94.6% and 96.9%, respectively), whereas sh777 repressed NeuroD1 by 74.8%. (B–H′′) Consequences of loss-of-function of NeuroD1 via in vivo postnatal electroporation at 4 and 15 dpe. (B–E) No differences were observed at the level of the lateral ventricle or in the RMS at 4 dpe. (F) Cell distribution along the rostro-caudal axis was normal (definition of levels in Fig. 2l). (G and H′′) Consequences of NeuroD1 knockdown on PGN morphology at 15 dpe. (G) Whereas shRNAs showing a strong effect on NeuroD1 expression strongly inhibited morphological differentiation, the weakly active shRNA 777 had only a minor effect compared with control. (H) Examples of cells that served for classification of PGN. Class1 cells present primary and secondary branching. Dendritic spines (arrowheads) indicate their synaptic integration in OB circuitry. Class 2 cells present a single primary branch. Class 3 cells present a spherical morphology and no branching. Errors bars indicate SEM. Statistics, unpaired t test. ns, not significant. **P < 0.01; ***P < 0.005. (Scale bar: 100 μm in B–E; 20 μm in H.
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When the two highly active NeuroD1-specific shRNAs sh775 and sh776 were electroporated, the vast majority of cells in the GL showed simple morphologies with few or no processes (classes 2 and 3), whereas cells with complex neuronal morphologies were sparse (Fig. 4G). When the less-efficient shRNA sh777 was expressed, an intermediate degree of neuronal maturation was observed (Fig. 4G), suggesting a dose-dependent action of NeuroD1 under these conditions. Comparable results were obtained for the GCL. As in the PGL, knockdown of NeuroD1 induced a dose-dependent inhibition of terminal neuronal differentiation (Fig. S9 A and B).
Thus, knockdown of NeuroD1 did not notably interfere with early steps of interneuron generation, but induced a specific defect in the acquisition of the differentiated neuronal phenotype in the OB.
Discussion
Although considerable information is available concerning the generation, specification, and migration of neurons, little is known concerning the factors and regulatory cascades that maintain the immature neuronal precursor status or induce the exit from this state and trigger terminal differentiation. Using a systematic approach, we identified NeuroD1 as a candidate for the latter function and validated this role using gain- and loss-of function approaches.
In Xenopus, a late function of NeuroD1 has been suggested based on two lines of evidence (13). First, NeuroD1 is transitorily expressed in territories where neuronal differentiation occurs. Second, misexpression of NeuroD1 causes the premature differentiation of neuronal precursors into neurons. However, the observation that NeuroD1 could also convert presumptive epidermal cells into neurons pointed toward a determination function. Therefore, a doubtless discrimination between a proneural and a terminal differentiation function was not possible.
The above-cited pioneering work in the frog has been extended through the analysis of mice with mutations in the NeuroD1 gene (14, 15, 21). In the hippocampal dentate gyrus of such animals, granule cell precursors are generated correctly in the neuroepithelium and invade the hippocampal anlage. However, in the target structure, precursors show a severe deficit in proliferation, and a defined dentate gryus is not formed (15). In the mutant cerebellum, generation and migration of early precursors appear not to be affected. Nevertheless, once these cells become postmitotic, massive cell death is observed and the cerebellum is severely affected (14). Thus, in these systems a late function of NeuroD1 is already suggested. However, because of the complexity of the models and the relatively low level of resolution, the available information is still fragmentary.
We attempted to clarify the role of NeuroD1 in neuronal differentiation by analyzing its function during olfactory neurogenesis. Using SAGE, microarray, in situ hybridization, and lacZ knockin into the NeuroD1 locus, we have demonstrated that NeuroD1 is expressed in mature neurons of the OB but is absent from immature stages. These findings are in contrast to recent expression data based on a NeuroD1 antibody, suggesting expression of the transcription factor already in the SVZ and RMS (23, 29). However, our loss-of-function approach based on RNAi shows that NeuroD1 is dispensable for generation and migration of precursors but is necessary for their transition into neurons in the target layer. These findings are in agreement with those of a recent study based on conditional NeuroD1 mutants, which showed a comparable defect in the OB (29).
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This work demonstrates that expression of a single transcription factor can induce massive ectopic neuronal differentiation of neural stem cells in the vertebrate forebrain. The existence of postnatal and adult neurogenesis holds potential for the treatment of neurodegenerative diseases (34). However, in many experimental paradigms, transplanted or recruited cells fail to undergo differentiation into neurons and either transdifferentiate into glia or remain immature precursors (18, 35). It appears conceivable to combine such approaches with the strong neuronal differentiation inducing activity of NeuroD1.
Scientists Unveil Critical Mechanism of Memory Formation
In a new study that could have implications for future drug discovery efforts for a number of neurodegenerative diseases, scientists from the Florida campus of The Scripps Research Institute (TSRI) have found that the interaction between a pair of brain proteins has a substantial and previously unrecognized effect on memory formation.
The study, which was published November 19, 2015 by the journal Cell, focuses on two receptors previously believed to be unrelated—one for the neurotransmitter dopamine, which is involved in learning and memory, reward-motivated behavior, motor control and other functions, and the other for the hormone ghrelin, which is known for regulating appetite as well as the distribution and use of energy.
“Our immediate question was, what is the ghrelin receptor doing in the brain since the natural ligand—ghrelin—for it is missing? What is it’s functional role?” said Roy Smith, chair of TSRI’s Department of Metabolism and Aging. “We found in animal models that when these two receptors interact, the ghrelin receptor changes the structure of the dopamine receptor and alters its signaling pathway.”
“This concept has potentially profound therapeutic implications,” said Andras Kern, the first author of the study and a staff scientist in the Smith lab, “pointing to a possible strategy for selective fine-tuning of dopamine signaling in neurons related to memory. By using small molecules binding to the ghrelin receptor we can enhance or inhibit dopamine signaling.”
Challenging the current theory, which involves canonical dopamine signaling in neurons, the new study shows that the biologically active ghrelin-dopamine receptor complex produces synaptic plasticity, the ability of the brain’s synapses (parts of nerve cells that communicate with other nerve cells) to grow and expand, the biological process underpinning long-term memory formation.
In addition, when the researchers blocked the ghrelin receptor, dopamine-dependent memory formation was inhibited in animal models, demonstrating the mechanism is essential to that process.
Combined with conclusions from earlier studies that showed a significant role for the ghrelin receptor in neurons that regulate food intake, insulin release and immune system deterioration due to aging, the new study further expands the ghrelin receptor’s importance. In animal models, ghrelin inhibits neuronal loss associated with Parkinson’s disease, and stroke, Smith noted, and the new study underlines its possible role in treating memory loss, age related or otherwise.
“All in all, it’s a pretty amazing receptor,” he said.
In addition to Smith and Kern, other authors of the study, “Hippocampal Dopamine/DRD1 Signaling Dependent on the Ghrelin Receptor,” are Maria Mavrikaki, Celine Ullrich, Rosie Albarran-Zeckler and Alicia Faruzzi Brantley of TSRI.
This work was supported by the National Institutes of Health (grant R01AG019230).
Hippocampal Dopamine/DRD1 Signaling Dependent on the Ghrelin Receptor
Andras Kern, Maria Mavrikaki3, Celine Ullrich4, Rosie Albarran-Zeckler, Alicia Faruzzi Brantley, Roy G. Smith
•In hippocampal neurons GHSR1a and DRD1 forms heteromers in a complex with Gαq
•DRD1-induced hippocampal synaptic plasticity is dependent on GHSR1a and Gαq
•DRD1 mediated learning and memory is dependent on Gαq-PLC rather than Gαs signaling
•DRD1-induced hippocampal memory is regulated by allosteric DRD1:GHSR1a interactions
The ghrelin receptor (GHSR1a) and dopamine receptor-1 (DRD1) are coexpressed in hippocampal neurons, yet ghrelin is undetectable in the hippocampus; therefore, we sought a function for apo-GHSR1a. Real-time single-molecule analysis on hippocampal neurons revealed dimerization between apo-GHSR1a and DRD1 that is enhanced by DRD1 agonism. In addition, proximity measurements support formation of preassembled apo-GHSR1a:DRD1:Gαqheteromeric complexes in hippocampal neurons. Activation by a DRD1 agonist produced non-canonical signal transduction via Gαq-PLC-IP3-Ca2+ at the expense of canonical DRD1 GαscAMP signaling to result in CaMKII activation, glutamate receptor exocytosis, synaptic reorganization, and expression of early markers of hippocampal synaptic plasticity. Remarkably, this pathway is blocked by genetic or pharmacological inactivation of GHSR1a. In mice, GHSR1a inactivation inhibits DRD1-mediated hippocampal behavior and memory. Our findings identify a previously unrecognized mechanism essential for DRD1 initiation of hippocampal synaptic plasticity that is dependent on GHSR1a, and independent of cAMP signaling.
This is a fascinating study. It is of considerable interest because it deals with several items that need to be addressed with respect to neurodevelopmental disruptive disorders. It leaves open some aspects that are known, but not subject to investigation in the experiments. Then there is also no reporting of some associations that are known at the time of deveopment of these disorders – autism spectrum, and schizophrenia. Of course, I don’t know how it would be possible to also look at prediction of a possible relationship to later development of mood disorders.
The placenta functions as an endocrine organ in the conversion of androsteinedione to testosterone during pregnancy, which is delivered to the fetus.
The conversion is by a known enzymatic pathway – and there is a sex difference in the depression of testosterone in males, females not affected.
There is a greater susceptibility of males to autism and schizophrenia than of females, which I as reader, had not known, but if this is true, it would lend some credence to a biological advantage to protect the females of animal species, and might raise some interest into what relationship it has to protecting multitasking for females.
It is well known that the twin studies that have been carried out determined that in identical twins, there is discordance as a rule. Those studies are old, and they did not examine whether the other identical twin might be anywhere on the autism spectrum disorder (not then termed “spectrum”.
However, there is a clear effect of stress on “gene expression”, and in this case we are looking at enzymation suppression at the placental level affecting trascriptional activity in the male fetus. The same genetic signature exists in the male genetic profile, so we are not looking at a clear somatic mutation in this study.
There is also much less specific an association with the MTHFR gene mutation at either one or two loci. This would have to be looked at as a possible separate post translational somatic mutation.
Whether there is another component expressed later in the function of the zinc metalloproteinase under stress in the affected subject is worth considering, but can’t be commented on with respect to the study.
Penn Team Links Placental Marker of Prenatal Stress to Neurodevelopmental Problems
By Ilene Schneider July 8, 2014
When a woman experiences a stressful event early in pregnancy, the risk that her child will develop autism spectrum disorders or schizophrenia increases. The way in which maternal stress is transmitted to the brain of the developing fetus, leading to these problems in neurodevelopment, is poorly understood.
New findings by University of Pennsylvania School of Veterinary Medicine scientists suggest that an enzyme found in the placenta is likely playing an important role. This enzyme, O-linked-N-acetylglucosamine transferase, or OGT, translates maternal stress into a reprogramming signal for the brain before birth. The study was supported by the National Institute of Mental Health.
“By manipulating this one gene, we were able to recapitulate many aspects of early prenatal stress,” said Tracy L. Bale, senior author on the paper and a professor in the Department of Animal Biology at Penn Vet. “OGT seems to be serving a role as the ‘canary in the coal mine,’ offering a readout of mom’s stress to change the baby’s developing brain. Bale, who also holds an appointment in the Department of Psychiatry, co-authored tha paper with postdoctoral researcher Christopher L. Howerton, for PNAS.
OGT is known to play a role in gene expression through chromatin remodeling, a process that makes some genes more or less available to be converted into proteins. In a study published last year in PNAS, Bale’s lab found that placentas from male mice pups had lower levels of OGT than those from female pups, and placentas from mothers that had been exposed to stress early in gestation had lower overall levels of OGT than placentas from the mothers’ unstressed counterparts.
“People think that the placenta only serves to promote blood flow between a mom and her baby, but that’s really not all it’s doing,” Bale said. “It’s a very dynamic endocrine tissue and it’s sex-specific, and we’ve shown that tampering with it can dramatically affect a baby’s developing brain.”
To elucidate how reduced levels of OGT might be transmitting signals through the placenta to a fetus, Bale and Howerton bred mice that partially or fully lacked OGT in the placenta. They then compared these transgenic mice to animals that had been subjected to mild stressors during early gestation, such as predator odor, unfamiliar objects or unusual noises, during the first week of their pregnancies.
The researchers performed a genome-wide search for genes that were affected by the altered levels of OGT and were also affected by exposure to early prenatal stress using a specific activational histone mark and found a broad swath of common gene expression patterns.
They chose to focus on one particular differentially regulated gene called Hsd17b3, which encodes an enzyme that converts androstenedione, a steroid hormone, to testosterone. The researchers found this gene to be particularly interesting in part because neurodevelopmental disorders such as autism and schizophrenia have strong gender biases, where they either predominantly affect males or present earlier in males.
Placentas associated with male mice pups born to stressed mothers had reduced levels of the enzyme Hsd17b3, and, as a result, had higher levels of androstenedione and lower levels of testosterone than normal mice.
“This could mean that, with early prenatal stress, males have less masculinization,” Bale said. “This is important because autism tends to be thought of as the brain in a hypermasculinized state, and schizophrenia is thought of as a hypomasculinized state. It makes sense that there is something about this process of testosterone synthesis that is being disrupted.”
Furthermore, the mice born to mothers with disrupted OGT looked like the offspring of stressed mothers in other ways. Although they were born at a normal weight, their growth slowed at weaning. Their body weight as adults was 10 to 20 percent lower than control mice.
Because of the key role that that the hypothalamus plays in controlling growth and many other critical survival functions, the Penn Vet researchers then screened the mouse genome for genes with differential expression in the hypothalamus, comparing normal mice, mice with reduced OGT and mice born to stressed mothers.
They identified several gene sets related to the structure and function of mitochrondria, the powerhouses of cells that are responsible for producing energy. And indeed, when compared by an enzymatic assay that examines mitochondria biogenesis, both the mice born to stressed mothers and mice born to mothers with reduced OGT had dramatically reduced mitochondrial function in their hypothalamus compared to normal mice. These studies were done in collaboration with Narayan Avadhani’s lab at Penn Vet. Such reduced function could explain why the growth patterns of mice appeared similar until weaning, at which point energy demands go up.
“If you have a really bad furnace you might be okay if temperatures are mild,” Bale said. “But, if it’s very cold, it can’t meet demand. It could be the same for these mice. If you’re in a litter close to your siblings and mom, you don’t need to produce a lot of heat, but once you wean you have an extra demand for producing heat. They’re just not keeping up.”
Bale points out that mitochondrial dysfunction in the brain has been reported in both schizophrenia and autism patients. In future work, Bale hopes to identify a suite of maternal plasma stress biomarkers that could signal an increased risk of neurodevelopmental disease for the baby.
“With that kind of a signature, we’d have a way to detect at-risk pregnancies and think about ways to intervene much earlier than waiting to look at the term placenta,” she said.
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