Paper 1 Flashcards
Describe/Explain the figure.
(A) – demonstrating the timeline of the administration of small molecules to the experimental culture (which ones, what days etc).
(B) and (C) – we can see atrocytic markers –> strocytes in the control culture. So starting culture was astrocytes, with no neurons already in the culture (growth/development of them must then be a result of the experimental conditions) – conclusion combined with D
(D) – very few neurons in the culture, because very little presence of tagged neuronal markers. Conclusion combined with B and C.
(E) and (F) – we see presence of fluorescently tagged neuronal markers, 14d and 30d after initial administration of small molecule treatment, which demonstrates that neurons are present in the experimental culture. In (F), the converted astrocytes (into neurons) have developed extensive dendrites (MAP2) and immunopositive for mature neuron marker NeuN. So, these converted astrocytes have survived 30 days, are developing dendrites and maturing.
(G) – 4months after initial small molecule treatement, presence of tagged neuronal markers, neurons still survive and developed dendritic trees and axons. So neurons are surviving, maturing, and integrating into neuronal networks.
(H) – Used astroglial lineage tracing to show that the astroglial cells, after treatment, have been converted into neurons (immunopositive for neuronal markers).
(I) and (J) – Demonstrates the efficiency of the MCM treatment, in comparing the immunopositivity of neuronal marker Tuj1 in control after 8 days versus experimental after 8 days.
(K) – showing efficiency of the treatement in converting human midbrain astrocytes to neurons (as opposed to cortical astrocytes, which is what they used primarily). So treatment works on both types of astrocytes.
(L) – the control of (K) condition, similarly showing that in the absense of treatement, very little presence of labelled neuronal markers.
(M) – graphical demonstration of (K) and (L), comparing presence of the mature neuronal marker NeuN+ cells in experimental versus control.
OVERALL: Sequential exposure to a defined grop of small molecules converts human astrolgical cells (both cortical astrocytes and human midbrain astrocytes) into neuronal cells.
Decribe/Explain the figure below.
A) Shows that small-molecule-induced human neurons are capable of long-term survival (in this case the figure shows them at 5 months). These cells are shown to have robust synaptic puncta (clusters of synaptic proteins labelled with antibodies) along the dendrites.
B-D) B and C are representative traces showing sodium and potassium currents at 1 and 2 months respectively (greater activity in the latter). D demonstrates that the current tapers down when the sodium currents are blocked by TTX.
E) Quantitative analysis of potassium and sodium current peaks in small-molecule-induced human neurons from 2 weeks to 2 months. We can see that the current increases dramatically in this time period, indicating that more and more astrocytes are being reprogrammed into neurons.
F) Representative trace of repetitive action potentials recorded in small-molecule-induced human neurons 75 days after initial treatment.
G-H) Representative trace showing all synaptic activity at 2 months post treatment. H is a zoom in of G that shows a close up of the synaptic activity.
I) It is revealed that these human astrocyte-converted neurons are exhibiting inhibitory GABAergic activity that is then blocked by GABA receptor antagonist BIC (at 2 months post treatment).
J-K) Representative trace showing synaptic burst activity in 3-month-old cells. K is a zoom in of J that shows a close up of this synaptic activity.
L) The burst activities were blocked by TTX (which blocks Na+ channels therefore preventing synaptic activity). Burst activities are also blocked by DNQX which is a glutamate receptor antagonist indicating that the burst activity were glutamatergic events.
M) This shows dual whole-cell recordings that demonstrate that the induced neurons developed robust synaptic networks and were capable of synchronous activity.
N) Calcium ratio imaging further demonstrated that the neurons were capable of synchronous activity and were highly connected (i.e., had robust synaptic networks).
OVERALL: This is a functional analysis that demonstrates that the human astrocytes have successfully been converted to neurons by small-molecule treatment because they show typical neuronal activity. Synchronous activity demonstrated –> astrocyte-converted neurons behaving as neurons.
Describe/explain the figure below.
(A-C) – Immunostaining shows that the astrocyte-converted neurons were positive for FoxG1, a forebrain marker, but not for hindrabin or spinal chord marker, indicated that they largely reprogrammed into forebrain neurons.
(D-F) – Immunostaining shows that the astrocyte-converted neurons were positive for deep layer marker, not corticial superficial layer marker, indicating that the chemically reprogrammed neurons are mainly deep layer forebrain neurons.
(G-H) – Immunostaining shows immunopositivity for general cortical neuron marker as well as hippocampal neuronal marker. Indicates that the chemically reprogrammed neurons are also hippicampal neurons.
(I) – shows presence of different markers they’re exploring.
(J) – converted neurons were immuno+ for glutamatergic neuron marker.
(K) – a few converted neurons were immuno+ for GABAergic neuron marker
(L-N) – Comparing immunopostivity for markers indicating neurotransmitter type. Largley immunonegative for VAChT(vesciular acetylcholine transporter) and TH(marker for dopamine neuron) or spinal motor neuron marker.
(O) – quantitative analyses of the nruonal subtypes. Suggesting that glutamatergic neurons are the major subtype resulting from the reprogramming.
OVERALL: Characterisation of the human astrocyte-converted neurons induced by small molecules. Comparing presence of different neuronal markers, to determine what kind of neurons have resulted from the reprogramming.
PROBLEMS: Have markers for forebrain glutamatergic neurons? Maybe a bit bold/lazy to thereby claim they are indeed those types of neurons. Also, didn’t ask about morphology.
Describe/explain figure below.
A-B) PCR array reveals that the converted cells have high levels of transcription for a number of neural transcription factors, i.e., NGN1/2 and NEUROD1, as well as the immature neuronal gene DCX at day 4 and 8 (at day 8 DCX increased >2000 fold compared to the control)
C-F) PCR analysis reveals the transcriptional changes that over time. Neural TFs increase and peak transcriptional activity occurs at day 4/6 while astrocyte TFs are downregulated significantly.
G-I) This demonstrates the epigenetic regulation of the GFAP (astroglial gene) promoter and transcription start site during chemical reprogramming. MeDIP-seq showed increased methylation of the GFAP promoter region after 8 days of treatment which was confirmed by BS-seq. The hypermethylated sites were located largely at 2 important transcription factor binding sites so methylation results in decreased transcription of GFAP. There is also increased methylation at the TSS at day 8 demonstrating that GFAP transcription if inhibited by DNA methylation.
J-K) MeDIP-seq and BS-seq show decreased methylation at day 8 compared to day 0 for NEFM, suggesting transcription activation of neuronal genes is occurring during chemical reprogramming.
L-M) Histone acetylation increases significantly in the NGN2 promoter region making DNA more available, resulting in transcription being upregulated.
N-O) Increased methylation of H3K4 and decreased methylation of H3K27, indicating that epigentic activation of NGN2 is occurring through histone modification.
OVERALL: Demonstrating that transcriptional and epigenetic regulation are involved in the chemical reprogramming of human astrocytes into neurons
Describe/explain the figure below
Attempting to corroborate with the transcirptional and epigenetic analyses. PErforming immunostaining to examine how protein expression changes during the reprogramming.
(A-C) – presence of 3 different neuronal transciption factors (TFs) from during different points of the treatment. Matching graphs (the quantitative analysis) (G–I). Peaks of presence at different points for the different TFs, expression of TF NeuroD1 delayed relative to the other 2.
Cells began to show neuronal marker DCX at D4-6, and the mature neuronal marker NeuN appeared D8-10, after peak expression of NeuroD1.
Astrocytic marker showed a decrease (F, K) –> epigenetic silencing and transcriptional downregulation.
OVERALL: Increased level of expression of neural TFs during the treatment. Suggests that the reprogramming treatment succesfully activated endogenous neural TFs, which may play a role in the reprogramming.
Describe/explain the figure below.
(A) – This is the control, treating human astrocytes with 1% DMSO, which is the medium the control cells are grown in. We can see that there is very little of the neural markers NeuN and MAP2 in the control (no treatment of small molecules).
(B) – This culture received the entire small molecule treatment, and imaging taken 14d after initial treatment. WE see the presence of the two neural markers, significantly greater in comparison with the control.
(C-F) – Here, various small molecules were individually removed from the MCM. We can see that the removal of certain small molecules had a great effect on the presence of neural markers, and some had less of an effect on neuronal conversion, if at all (proxy is the presence of the neural markers in the culture).
(K) – Shows the quantitative analyses, demonstrating that removing DAPT had the most significant impact on reprogramming efficiency, followed by others. Removal of two of the small molecules appeared to have no significant effect on the neuronal conversion, indicating that two of the 9 small molecules are shown to be unnecessary. (One of them, TTNPB was included because is an agonit of RA receptors, found to play an important role in nueral differentiation, but this result suggests that it may not be essential to astrocyte-neuron reprogramming).
OVERALL: Determining which of the small molecules are essential to the neuronal reprogramming; evaluating the conversion of astrocytes to neurons as they take each molecule out one by one.
PROBLEMS: Counfound – only at figure 6 do they show that all molecules are necessary, and in fact 2 are shown to be unnecessary.
Describe/explain the figure below.
(A) – Infected the human astrocytes with a virus labelled with EGFP so that these astroctyes and astrocyte-converted neurons (after treatment with MCM) would be labelled and distinguishable from native mice neurons/astrocytes. Injection took place in the lateral ventricle of neonatal mice.
(B) – 7 days post injection, we see GFP+ cells which were positive for HuNu, human nuclei (suggesting these cells originated from injection of human cells). These were also positive for neuronal markers DCX, MAP2, and mature neuronal marker NeuN (B–D). –> Suggests that human astrocyte-converted neurons can survive in the mouse brain in vivo.
(D) – some GFP+ human neurons also positive for NeuN and HuNU can be seen in adjacent striatum areas, so migrated to adjacent regions of injection.
(E) – “ “ , can be seen in brain areas adjacent to the injection area in the thalamus and striatum – these human reprogrammed neurons may have migrated out of the injection area and integrated into the local neural circuits.
(F) – supports above statement, presence of synpatic puncta (SV2) along dendrites of the EGFP+ human neurons –> may have established synpatic potentials with host neurons.
OVERALL: The human astrocyte-converted neurons appear to be able to survive in vivo in a mouse, and survive and integrate in the neural circuitry of that mouse brain.
