All lectures

Question 1

1.1. [structure of chemical synapses] Name or indicate structures on images of synapses

Answer 1

take out image and do that

Question 2

1.2. [structure of chemical synapses] How are synapses classified based on (a) type of NT, (b) postsynaptic receptors, postsynaptic responses, and (c) ultrastructural morphological features

Answer 2

a. chemical synapses can be excitatory (e.g. glutamate), inhibitory (e.g. gaba or glycine) or neuromodulatory (e.g. monoamines).
Furthermore, synaptic diversity is also based on their location (central vs peripheral) - for example the neuromuscular junction, the neuro-endocrine junction)
b. Excitatory synapses depolarize the post synapse (ampa and nmda receptors), while inhibitory synapses hyperpolarize it. As for neuromodulatory synapses they do not have ionotropic receptors, they induce biochemical changes in the postsynapse
c. The ultrastructural morphological features of synapses include: central synapse (where the axon of the presynapse contacts the dendrite of a postsynaptic neuron), type 1 (symmetrical) - usually excitatory and on dendrides and dendritic spines, type 2 (asymmetrical) usually inhibitory and on soma and axonal initial segment . On the presynapse plasma membrane we can find the active zone, a specialized region where vesicles are docked and primed for release, the AZ is aligned with the post synaptic density. The molecular composition of PSD includes: 1. neurotransmitter receptors 2. transsynaptic adhesion molecules 3. scaffolding molecules 4. signal transduction molecules.

Question 3

2.1. [SCAMS] What are the functions of SCAMs at synapses?

Answer 3

Scams (synaptic cell adhesion molecules) are synaptic junctions organized by trans-synaptic cell-adhesin molecules bridging the synaptic cleft. Apart from connecting pre and post-synapses they also mediate trans-synaptic recognition and signaling processes that are essential for the establishment, specification, and plasticity of synapses.

Question 4

2.2. [SCAMS] Define the role of SCAMs during synaptic formation stages (describe the stages as well) and synaptic function

Answer 4

Scams contribute to synaptic formation and function.
Stage 1. contact establishment: here pre and post-synapse establish contact through homophilic and heterophilic interactions between SCAMS to recognize the appropriate synaptic partners

Stage 2. recruitment of synaptic vesicles: once contact is established, synaptic vesicles are recruited. Here SCAMs regulate physical cell-cell adhesion and serve as anchor proteins to cluster or recruit receptors or components of the pre-and post-synaptic signaling machinery

stage 3. functional specification: molecular components of synapses are organized resulting in the functionality of the synapse
stage 4. synaptic plasticity: SCAMs may contribute to structural and functional changes in activity-dependent adaptive events (plasticity)

Question 5

2.3. [SCAMS] What are the examples of SCAMs and their functional domains?

Answer 5

Adhesive function of scams is based on a limited number of extracellular domains , often assembled into repeat units.

SCAMs domains are
1- lamin A, neurexins (alpha and beta) and sex hormone binding protein (LNS)
2- neuroligins
both implicated in
schizophrenia and autism
3- immunoglobulin (lg)-domain proteins (e.g. synCAM) - usually homophilic, usually also contain fibronectin III domains - both heterophilic and homophilic
4- receptor phosphotyrosine kinases (phosphorylate) and phosphatases (dephosphorylates)
5- leucine-rich repeat proteins (LRR)
6-cadherin domains - always occur in multiple copies connected by a linker that binds 2-3 ca2+ ions. Usually homophilic.

Cadherins:
* influence early synapse development and impact synaptic plasticity
contain 5 extracellular cadherin repeat domains (EC1-5), with the N-terminal EC1 domain mediating adhesion in trans
* e.g. N-cadherin modulate synaptic function

NCAM: regulate synaptic plasticity

SynCAMs: synaptic cell adhesion molecules: organize excitatory synases
* contain 3 extracellular IgG domains, a single transmembrane region, and intracellular FERM- and PDZ-domain-binding motifs

SALMs: synaptic cell adhesion-like molecules: cluster post-synaptic plasticity
* single-pass membrane proteins with N-terminal LRR domain, a single Ig domain and fibronectin IlI domain, a transmembrane region and a cytoplasmic tail
* vertebrates contain 5 SALM genes

Question 6

2.4. [SCAMS] draw SCAMs to illustrate how they function

Answer 6

see figures.
Functions of SCAMs have been discovered thanks to knock out animal models. Rodents wit the KO alpha Nrx suggest that alpha neurexin and neuroligin coordinate the recruitment of ca2+ channels and components of the release machinery, They also show reduced neurotransmitter release and that neurexin ligands do not act as synaptic glues but as activity-dependent regulators of synapse function - if disrupted they’re not essential for synaptic transmission however they affect its efficacy (e.g- less nt release probability, less ca2+)

Cadherins:
* influence early synapse development and impact synaptic plasticity
contain 5 extracellular cadherin repeat domains (EC1-5), with the N-terminal EC1 domain mediating adhesion in trans
* e.g. N-cadherin modulate synaptic function

NCAM: regulate synaptic plasticity

SynCAMs: synaptic cell adhesion molecules: organize excitatory synases
* contain 3 extracellular IgG domains, a single transmembrane region, and intracellular FERM- and PDZ-domain-binding motifs

SALMs: synaptic cell adhesion-like molecules: cluster post-synaptic plasticity
* single-pass membrane proteins with N-terminal LRR domain, a single Ig domain and fibronectin IlI domain, a transmembrane region and a cytoplasmic tail
* vertebrates contain 5 SALM genes

Question 7

2.5 [SCAMS] Make a schematic drawing to illustrate neurexin-neuroligin interactions. Indicate and name the different functional domains.
How are these type of molecules called? What is the function of neurexin-neuroligin interactions?
-After answering, still look at the two slides containing info - not everything is written in the answer

Answer 7

Schematic drawing: slide 19. Neurexins and neuroligins are synaptic cell adhesion molecules.
Functional domains: extracellularly, a-neurexins contain 6 LNS (Lamin A, neurexin and sex-hormone-binding protein domains) domains with 3 interspersed EGF-like domains;
beta-neurexins only contain a single LNS domain. Intracellularly, the short cytoplasmic tails of neurexins contain PDZ-domain binding sequences that bind to intracellular proteins.
Neuroligins: their extracellular sequence contains an esterase-like domain that forms a constitutive dimer. Cytoplasmic neuroligin tails contain a PDZ-domain-binding sequences (and a tyrosine-based motif)
Neurexins bind to neuroligins to form trans-synaptic cell adhesion complexes, using the sixth LNS domain of a-neurexin and the single LNS domain of 6-neurexin.
Neurexin-neuroligin interactions are thought to have vital functions in organizing synapses, e.g. recruitment of calcium channels and components of the release machinery to presynaptic active zones.

Question 8

3.1. [exocytosis] What are SNAREs? which snares are involved in vesicle exocytosis?

Answer 8

SNARE proteins mediate vesicle fusion
- SNARE proteins: proteins containing a SNARE domain

• SNARE= soluble NSF (N-ethylmaleimide sensitive factor)-attachment protein receptor
  SNARE proteins are targets of clostridial botulinum and tetanus toxins; these neurotoxins enter the presynaptic terminal and act as highly specific proteases, leacing to a selective block of presynaptic membrane fusion

Three SNARE proteins are essential for SV fusion:
* the vesicular SNARE protein synaptobrevin/VAMP (vesicle-associated membrane protein)
* SNAP-25
* syntaxin-1
* The synaptic SNARE complex (synaptobrevin-SNAP-25-syntaxin-1)
forms a parallel four-helix bundle

Question 9

3.2. [exocytosis] draw the functional domains of snares involved in sv exocytosis and the assembled snare complex

Answer 9

see slides + figure.

• SNARE proteins contain a SNARE motif, a characteristic sequence of 70-80 residues
  -the linker sequence of SNAP-25
  serves for membrane anchoring via palmitoylation
  palmitoylation: covalent attachment of fatty acids, e.g. palmitic acid, to cysteine (and less frequently to serine or threonine) residues of proteins.
• the energy released during Synaptic SNARE complex assembly fuels membrane fusion, likely by a simple mechanical force

Question 10

3.3. [exocytosis] describe SNARE/SM protein cycle and draw it

Answer 10

see figure

1. assembly/priming - chaperones: CSPalpha/beta/gamma+ alpha/beta/gamma synucleins - N to C terminal zippering of trans-snare complexes
2. fusion pore opening- partial trans snare proteins / sm protein assembly
3. fusion pore expansion- trans-snare proteins are converted onto cis-snare complexes (i.e. snare complexes on a single membrane)
4. recycling - snare complex disassembly and vesicle recycling - ATPase NSF and SNAPs dissociate cis-snare complexes into monomers. SNF is also the ATPase that loads snare proteins with energy

chaperones - proteins that assist the confirmation folding/unfolding of proteins as well as the assembly/disassembly of multi protein complexes

sm proteins

-evolutionary conserved cytosolic proteins; essential partners for SNARE proteins in fusion
-The N-terminus of syntaxin-1 tethers the SM protein Munc18, and this interaction is absolutely essential for fusion in vivo
-SNARE- and SM-protein complexes may stabilize the attachment of vesicles to the target membrane, thereby participating in docking

Question 11

3.4. [exocytosis] how do chaperones support snare protein function? draw it as well

Answer 11

check figure

Chaperones are proteins that assist the confirmational folding/unfolding of proteins as well as the assembly or disassembly of multi-protein complexes

Two types of chaperones support SNARE protein function

1. the classical chaperone complex SPa (cysteine string protein a, a SV protein), Hsc70, and SGT (small glutamine-rich tetratricopeptide repeat protein). This complex binds to SNAP-25 on the target membrane, thereby supporting the functional competence of SNAP-25 to engage in SNARE complexes.
2. the nonclassical chaperones a/B/y-synucleins, which are bound to phospholipids and synaptobrevin/VAMP on vesicles, and bind to assembling SNARE complexes to support their folding.

Question 12

3.5 [exocytosis] how do dysfunctional chaperones result in neurodegeneration? Draw it.

Answer 12

Defective chaperone function is implicated in neurodegeneration
* Both a-synuclein and CSPa are linked to neurodegeneration:
* a-synuclein mutation or duplication causes familial Parkinson’s disease
* many neurodegenerative diseases feature Levy bodies, which contain a-synuclein (e.g. Parkinson’s disease, Lewy body dementia)
* deletion of CSPa in mice has no immediate effect on neurotransmitter release, but leads to increased ubiquitination and degradation of SNAP-25 and to reduced SNARE-complex assembly, resulting in fulminant neurodegeneration that kills mice after 2-3 months
- abnormal exposure of neurons to misfolded SNAREs and/or abnormal SNARE complex assembly impairs neuronal survival

UBIQUITINIFICATION = covalent attachment of ubiquitin to proteins that need to be degraded

Question 13

4.1 [calcium control] Explain mechanisms by which ca2+ controls nt release

Answer 13

draw the timescale of neurotransmission and check the image

only local increase of ca2+ levels at pe-synapse trigger synchronized release
- we focus on how ca2+ triggers exocytosis

Ca2+ binding to synaptotagmin triggers synaptic vesicle exocytosis

How it goes in pre-synapse:
> action potential opening of Ca2+ channels transient increase in local Ca2+ concentration
> Ca2+ binding to synaptotagmin via two C2-domains
> interaction of synaptotagmin C2 domains with phospholipids and SNARE proteins
activation of the membrane fusion machinery

> In triggering exocytosis, synaptotagmins require an obligatory cofactor called complexin, a small protein that binds to SNARE proteins and simultaneously activates and clamps the SNARE complex for subsequent synaptotagmin action

> This mechanism operates in most, if not all Ca2+ regulated forms of exocytosis throughout the body, including degranulation of mast cells, acrosome exocytosis in sperm cells, hormone secretion from endocrine cells, and neuropeptide release

Question 14

4.2. [calcium control] describe the structure of the three groups of synaptotagmins and draw it

Answer 14

check drawing.

16 genes encoding canonical Syts are expressed in mammals
* Synaptotagmins (Syts) contain a short N-terminal sequence, transmembrane region, a central linker sequence, and two C-terminal C2-domains
* C2-domains: Ca?+ binding domains found in a large number of signal transduction and C membrane trafficking proteins
* Syt1, Syt2, Syt9 and Syt12 are expressed on synaptic vesicles
* Not all C2-domains bind Ca2+
* Syts are classified in two groups:
Ca2+-dependent: further subclassified based on the presence/absence of disulfide-bonded cystein residues in N-terminus

> Both C2-domains of Syt1 bind to phospholipids in a Ca2+-dependent manner, and to SNARE proteins:
* Syt1 binding to phospholipids requires Ca2+ and negatively charged phospholipids, with phosphatidyl-inositol phosphates being most effective
* the Syt1 C2-domains seem to directly interact with syntaxin-1; this interaction is greatly enhanced by Ca2+
* Syt1 also binds to assembled SNARE complexes in a Ca2+-dependent manner

Question 15

4.3. [calcium control] which synaptotagmins function as synaptic ca2+ sensors for NT release (a) and how this has been discovered and demonstrated (b)

Answer 15

syt1,syt2,syt9 function as synaptic Ca2+ sensors for nt release

A. Deletion of Syt1 in cortical neurons blocks fast synchronous NT release; note that not all Ca2+ stimulated release was abolished, a delayed asynchronous form of release is retained; release induced by hypertonic sucrose (thought to cause Ca2+-independent exocytosis of all vesicles in the readily releasable pool) is unchanged
-> Syt1 KO did not interfere with vesicle fusion as such, only with the Ca2+ triggering of fusion.

B. A systematic screen of all Ca2+-binding Syts for rescue of the Syt1 KO phenotype revealed that only Syt1, Syt2 and Syt9 were able to rescue

C. Syt1, Syt2, and Syt9 mediate Ca2+ triggering of release with distinct kinetics: Syt2 exhibits the fastest rise and decay kinetics, whereas Syt9-mediated IPSCs are two-fold slower. This fits well with Syt2 primarily expressed in synapses requiring very fast transmission (e.g. auditory system or NMJ) and Syt9 primarily expressed in the limbic system

D. Quantification of increase and decay of the IPSCs

syt1 specifically
Syt1 Ca2+-binding point mutations in the C2A-domain:
* D232N: increases the amount of Ca2+-stimulated SNARE complex binding, without altering phospholipid binding
* R233Q: greatly decreases the apparent Ca2+ affinity of Syt1 during phospholipid binding, without altering SNARE complex binding
* D238N: modestly decreases the apparent Ca2+ affinity of Syt1
> Conclusions:
* Svt1 is a true Ca2+ sensor
* Both SNARE- and phospholipid-binding by Syt1 are involved in release

Question 16

4.4. [calcium control] explain and draw how complexin and synaptotagmin function together to mediate ca2+-induced synaptic vesicle exocytosis.

Answer 16

what are Complexins?
> small soluble proteins (=120 amino acids), evolutionary conserved in mammals, which bind to the partially assembled SNARE complex
> Four complexin isoforms in mammals, with complexin-1 and -2 widely distributed in the body and abundant in the brain
> Deletion of complexin-1 and -2 in mice induces a milder phenocopy of the Syt1 KO phenotype (e.g. partial loss of synchronous NT release).

mechanism of action of synaptotagmin and complexion in Ca2+ triggered exocytosis
Current model: complexin binding to primed SVs containing partially assembled SNARE complexes “superprimes” the SV into an activated state, and subsequently clamps them

Ca2+ binding to Syt then triggers Syt binding to the SNARE complex and the phospholipid bilayer, dislodging the complexin clamp and pulling on the SNARE complex, thereby opening the fusion pore

Priming: partial SNARE/SM protein complex assembly

Superpriming: binding of complexin to partially assembled SNARE complexes
Fusion pore opening: triggered by Ca?+ binding to Syt

summary
* Complexins act both as activators and as clamps of NT release
* SNARE complex binding by complexin is essential for its function, and the complexin N-terminus is crucial for its activating role
* How does complexin act to promote Ca2+ triggering of SV fusion?
- the central a-helix of complexin and Syt1 bind to SNARE complexes at overlapping sites
- Ca2+ binding to Syt1 triggers displacement of complexin from the SNARE complex

Question 17

5.1. [endocytosis] what are the different endocytic pathways that can be used for synaptic recycling?

Answer 17

reproduce image and check it.

1. endocytosis of sv via clathrin-coated pits (CCPs) from the plasma membrane and its deep unfolding
   in other words: after sv is formed ccps are being released. This leads to acidification of sv and nt will be pumped in sv
2. kiss and run (still unsure if it happens)
   in other words: sv only partially fuse with the pre synaptic membrane and the fusion pore is briefly opened and closed
   ———more on it:
   > “kiss and run”: this mechanism is debated/controversial; only indirect evidence exists
3. bulk endocytosis by sv formation from endocytic intermediates (EI)
   in other words: endocytosis of larger parts of the pre-synaptic membrane leads to the formation of EI (potentially sv could be regenerated through this mechanism) - it can be either clathrin-dependent or independent
   —— more on it: Bulk endocytosis
   > operates most prominently under strong stimulatory conditions, when a large number of synaptic vesicles fuse with the plasma membrane within a short time interval
   > excess plasma membrane is rapidly captured via the formation of plasma membrane infoldings, which then undergo fission to generate intracellular vacuoles and cisternae (endosome-like intermediates, El)
   > this is a non-selective mechanism of membrane uptake, but the resulting Els may be enriched with intrinsic SV membrane proteins.
   These Els subsequently give rise to new SVS
   > The molecular mechanisms of bulk endocytosis are largely unknown:
   * the fission of deep membrane infoldings is dynamin-independent
   * the actin cytoskeleton and proteins that couple membrane deformation to actin dynamics may be involved e.g. the F-BAR protein syndapin
   ——————–
4. housekeeping membrane recycling involving clathrin-mediated endocytosis and canonical early endosomes (EE), as well as traffic to the cell body via late endosomes (LE) and multivesicular bodies (MVB)
   in other words: housekeeping membrane recycling involves clathrin endocytosis, which leads to the formation of EE which can then be converted into LE that can travel retrogradely to the cell’s cell body

———— more on it: > nerve terminal endosomes: the role of “canonical” early endosomes (organelles downstream of clathrin-coated vesicles and other vesicles that form directly from the plasma membrane) remains a poorly explored topic

Question 18

5.2. [endocytosis] what are the methods to study sv endocytosis? Describe them and infer which method is best suited for a given experimental condition

Answer 18

1. Amphiphatic styryl dyes such as FM1-43. These dyes have an apolar side which results in their binding to membranes, and a polar side which prevents the molecules to pass through membranes. These molecules are phluorescent, allowing their visualization, and the phluorescence intensity can be quantified. Incubation of cultured cells, Drosophila NMJs or other synaptic systems with FM1-43 results in binding of FM1-43 to membranes (including presynaptic membranes). Stimulation of vesicle exocytosis, e.g. by incubation with high potassium buffers, results in exocytosis and subsequent endocytosis of synaptic vesicles, resulting in uptake of FM1-43 in synaptic vesicles. After a subsequent washing step, only FM1-43 that has been taken up in SVs will remain, and the fluorscence intensity of presynatpic terminals is a proxy for the number of endocytosed SVs.
2. SynaptopHluorin: a fusion protein of pHluorin to the lumenal portion of synaptic
   vesicle proteins (typically synaptobrevin). pHluorin is a GFP variant (pKa = 7.1), of
   which the fluorescence is quenched at the acidic pH of SVs, and recovered on SV fusion and exposure to the near-neutral extracellular pH. Thus, stimulation of exocytosis, e.g. by high-frequency stimulation of cultured neurons, will result in an increase of pHluorin fluorescence, followed by a decrease which is due to SV recycling by endocytosis, followed by re-acidification.
   Application of bafilomycin, a membrane-permeable blocker of the V-type ATPase that is required for vesicle re-acidification, can be used to trap vesicles at neutral pH after endocytosis, thus allowing synaptopHluorin to remain fluorescent even after endocytosis. Thus, comparing the change in fluorescence intensity in presynaptic terminals upon HFS in the presence and the absence of bafilomycin allows to estimate total endocytosis (difference between the two traces).
3. electrophysiology: endocytic recovery of the increase in surface area produced by a secretory burst at the calyx of Held giant nerve terminal can be monitored by measurement of membrane capacitance (Cm).
4. Imaging methods, e.g. internalization of antibodies against luminal domains of SV proteins. Antibodies can either be fluorescently labeled or coupled with the pH-sensitive cyanine dye derivate Cy-pHer5E.

Question 19

5.3. [endocytosis] discuss the sequential steps of clathrin-mediated endocytosis (a), the different classes of molecules involved, and give examples of molecules (b).

Answer 19

draw and then check figure on slide.

A

Clathrin-mediated endocytosis
> Major pathway of endocytosis, in particular at rest and during modest activity

> Steps in the nucleation of clathrin-coated pits:
step1. interaction of clathrin adaptors and/or a subset of their accessory factors with the lipid bilayer and with membrane proteins
step2. interactors of adaptors with each other, with other accessory clathrin
factors, with cargo proteins, and with clathrin adaptor leads to the rapid growth of the coat in a feed-forward, cooperative fashion.
step3. a deeply invaginated clathrin-coated bud with a narrow neck is formed; fission of this neck in a reaction that requires the GTPase dynamin leads to a free vesicle that rapidly uncoats.

> Unique properties of clathrin-mediated SV endocytosis:
* highly homogeneous small size of the vesicles (there must be molecules that make sure vesicles have the right diameter when endocitosed)
* specificity of the cargo: all needed membrane proteins have to be included into the nascent vesicle in the appropriate stochiometry
* speed of the process (15-20 sec)

> Molecular mechanisms underlying this specificity: poorly understood

> The clathrin lattice is a chicken-wire-like structure, likely involved in generation/maintenance of membrane curvature. Clathrin also functions as a scaffold for the clustering of the adaptors and thus of the membrane cargo to be internalized.

> adaptors bind to:
1. cytoplasmically exposed domains or endocytic “motifs” of vesicle membrane proteins, as well as to PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane
2. clathrin heavy chain and/or other adaptors and endocytic factors

B
Major endocytic proteins involved in sv recycling include
clathrin coat components (Clathrin, AP-2, epsin - PI(4,5)P2, stonin)
BAR domain- containing components (endophilin, amphiphysin)
phosphoinositide metabolism (pipk1 gamma, synaptojanin)
scaffolding (intersecting, eps15)
membrane fission (dynamil)
clathrin lattice dissembly (hsc70 auxilin)

• Interactions are important to recruit and concentrating all the synaptic vesicles proteins that need to be recycled and bring them closely together

The adapter proteins interact, not only with the synaptic vesicles proteins (eg AP 2 with synaptojanin) but also with other proteins (eg PI (4,5) P2)

Bar domain containing proteins
A. ,generate/sense bilayer curvature
B. interact with plasma membrane which generates membrane curvature because it likes curved membranes

Clathrin coated pits are typically observed at the outer margins of the active zones (=endocytoc or periactive zone)

Question 20

5.4. [endocytosis] how do membrane fission and uncoating occur?

Answer 20

• membrane fission requires the GTPase dynamin: dynamin oligomerizes into spirals at bud necks –> GTP-dependent dimerization of the GTPase module –> GTP hydrolysis –> conformational change of neighboring domains –> constriction the dynamin spiral and the underlying tubular bud neck
• Uncoating (clathrin shedding) is ATP-dependent and requires Hsc70
  ATPase and its cofactor auxilin
• Shedding of adaptors is dependent on
  PI(4,5)P2 hydrolysis by synaptojanin

Question 21

6.1 [sv pools] what is the 3 pool model (a)? how does it function (b)? Describe it

Answer 21

• The existance of distinct SV pools was first suggested by electrophysiological experiments: during high-frequency stimulation of presynaptic neurons, the postsynaptic response declined over time = synaptic depression - so the frequency of psr decreases over time - how can it be explained?

Hypothesis: during frequent stimulation, NT quanta are released from a limited pool of “release-ready” SVs, emptying the pool at a rate faster than fresh SVs could replenish those expended.
> Assuming that each AP discharges a similar fraction of SVs, a progressive decrease in release is expected with each stimulus, until a lower steady state level, in which release is perfectly balanced by the slow refilling of the “readily-releasable pool”.
Thus, only a limited fraction of SVs is “release-ready” and additional SVs are recruited to replenish this pool. - It takes longer to refill the docking zone with vesicle that the “BREAK” between stimulations

Readily releasable pool (RRP): consists of SVs that are release-ready
Recycling pool (RP): consists of SVs that can replenish the RRP
> Total recycling pool (TRP)= RRP + RP: groups all SVs capable of undergoing release
Resting pool (R,P): consists of SVs that remain unreleased, even after prolonged stimulation

Question 22

6.2 [sv pools] explain the methods to study sv pools and apply to a given scientific question

Answer 22

methods to distinguish RTP from RtP

1. > TRP can be labeled using strong stimulation in the presence of extracellular FM dye or quantum dots (QDs).
   QDs are nanoparticles of uniform and defined size, which have remarkably stable and bright photoluminescence (or electrodense core).
   To quantify the number of SVs in the TRP, these methods can be combined with EM, using photoconversion. In this procedure FM dye-labeled synapses can be exposed to the chemical 3,3’-diaminobenzidine (DAB), and prolonged photoillumination causes excitation-induced conversion of DAB to an electron-dense product only in dye-containing SVs (exocytosed and then endocytosed- trp pool). Also QDs with an electron-dense core can be used.
   - allows to count vesicles

2
> PHIuorin: GFP variant (pK, = 7.1): fluorescence is quenched at the acidic pH of SVs, and recovered on SV fusion and exposure to the near-neutral extracellular pH - sv have a 3-4 ph, so fluorine doesn’t fluoress but then if exocytosed they do.
> SynaptopHluorin: fusion protein of pHluorin to the lumenal portion of SV proteins.
> upon high-frequency stimulation, all SVs from the TRP will be released. - stimulating the presynaptic nerve (10hz) leads to exocytosis and fluorescence
> Acute application of bafilomycin (BAF) prevents re-acidification of recycled vesicles, thus resulting in fluorescent labeling of these SVs - if we add baf sv will fluoress when exocytosed but if endocytosed again then baf makes sure that sv can’t be re-acidified so sv will keep fluoressing
> Ammonium chloride (NH4CI) neutralizes the acidic interior of R,P vesicles, resulting in unquenching of synaptopHluorin in these SVs - NH4cl makes sure that ph of sv will be neutralized (fluorescence starts)
> measurement of the change in fluorescence intensity during HFS with
BAF and subsequent NH,CI thus allows to identify TRP and R,P
- doesn’t allow to count vesicles

3
> The number of SVs in the RRP (NRRp) can be determined using electrophysiological methods, but also using an osmotic challenge, typically hypertonic sucrose, which induces Ca?+-independent exocytosis of SVs in the RRP

4
> Other tools and methods often used in the study of SV pools: cf. Box 1 in Alabi and Tsien, Cold Spring Harb Perspect Biol, 2012

Question 23

7.1. [vesicular and plasma membrane nt transporters] name them

Answer 23

SV transporters for NTs (SVNTTs)
> SVs fill with NT through a process driven by the vacuolar-type H-ATPase, which uses the energy released by ATP hydrolysis to pump protons in the SV lumen
> there are 3 major determinants of SV filling with NT:
* cytosolic concentration of the NT
* electrochemical driving force across the SV membrane (determined by the activity of the vacuolar-type H-ATPase)
* intrinsic properties of the SVNTT

classical SVNTTs:
VGLUT (1-3): vesicular glutamate transporter: exchanges H* for glutamate
VMAT: vesicular monoamine transporter: recognizes multiple monoamines as substrates; exhanges 2 lumenal H* for one protonated monoamine
VAchT: vesicular acetylcholine transporter: exchanges H* for Ach
VGAT: vesicular GABA transporter: recognizes both inhibitory NTs
GABA and glycine; co-transports GABA and 2 Cl

• monoamine NTs include dopamine, noradrenaline, adrenaline and serotonin

Question 24

7.2. [vesicular and plasma membrane nt transporters] explain their function (how) and include their driving forces and their role in nt cycles

Answer 24

Vesicular and plasma membrane NT transporters

on SVs:
* load SVs with NT
or
on plasma membrane:
* terminate synaptic transmission
* recycle NT

1-SV exocytosis leads to secretion of acetylcholine (ACh) into the synaptic cleft, allowing ACh to activate postsynaptic ACh receptors (AChR).
2-ACh in the synaptic cleft is hydrolized by acetylcholinesterase (AChE), resulting in the production of acetate and choline (Ch).
3-choline is taken up in the presynaptic neuron by the high-affinity choline transporter (ChT).
4-in the presynaptic nerve terminal, choline is converted into ACh again by choline acetyl transferase (ChAT).
5-ACh is subsequently transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT).

example for glu
glutamine-glutamate cycle
> Glutamate is taken up by astrocytes using the excitatory amino acid transporters 1 and 2 (EAAT1, 2).
> In astrocytes, glutamate is converted into glutamine by glutamine synthase
> Glutamine is transferred back to neurons through system N transporters expressed by glia and system A transporters expressed by neurons. System N transporters (SN1, 2) exchange 1 Na* and 1 glutamine for 1 H. System A transporters (SA1-3) couple the movement of neutral amino acids (e.g. glutamine) to the flux of Na.
> Within neurons, glutamine is converted to glutamate (and ammonia) by phosphate-activated glutaminase (PAG).
> NT is also transported back into the presynaptic terminal by Na* and
CI-dependent plasma membrane transporters
(exproins previous suite)

Question 25

7.3. [vesicular and plasma membrane nt transporters] infer how they can modulate synaptic strenght

Answer 25

> regulation of NT transporter activity contributes to synaptic plasticity:
* increased activity of SVNTTs -
-> increased NT concentration in
SVs, and simultaneously reduced NT concentration in the cytoplasm
* reduced NT uptake at the plasma membrane increases postsynaptic receptor activation but depletes NT stores

Question 26

7.4. [vesicular and plasma membrane nt transporters] understand/explain the effects of drugs that act on the plasma membrane nt transporters (PMNTTs) -a- and how genetic mutations in PMNTTs may lead to disease -b-

Answer 26

How is activity of PMNTTs regulated?
> gene expression regulation: regulation of transcription and splicing of pR
> trafficking: PMNTTs undergo both constitutive and regulated trafficking to and from the cell surface, a process influenced by multiple kinases (e.g. PKC, CaMKII, MAPK, ERK, PKG).
> Phosphorylation-dependent catalytic activation
> PMNTT-associated proteins:
* SNARE proteins, in particular syntaxin 1A (SYN1A): association of SYN1A with PMNTTs typically inhibits PMNTT activity
* kinases and phosphatases, e.g. PKC, PKG, CaMKIl, protein phosphatase 2A (PP2A)
* cell surface receptors, e.g. integrinß3-containing adhesion receptors (ITGB3), D2 DA receptors which associate with DAT, leading to enhanced DAT surface expression and DA uptake

Diseases linked to PMNTT dysfunction
> A457P mutation in NET: leads to orthostatic intolerance (Ol), a.k.a. postural orthostatic tachycardia syndrome (POTS), characterized by syncope on standing. The A457P mutation strongly reduces NET surface expression and hence NE uptake. Interestingly, Ol subjects also show cognitive impairments, presumably due to reduced NET activity and increased NE signaling in the brain
> a promoter variant in the NET gene that results in recruitment of a transcriptional repressor leading to reduced NET expression, influences the risk for ADHD, as well as systolic blood pressure
> genetic variation in SERT contributes to obsessive-compulsive disorder (OCD) and autism-spectrum disorders (ASD)
> A559V variant in DAT is linked to ADHD and bipolar disorder

Question 27

7.5. [vesicular and plasma membrane nt transporters] sv vs plasma membrane transporters

Answer 27

——————-SV transporters for nt (SVNTTs)
> cytosolic NT concentrations depend on:
* NT biosynthesis by specific enzymes: can be rate-limiting, e.g. tyrosine hydroxylase (TH) in catecholamine biosynthesis. TH activity is regulated by phosphorylation and regulation at the transcriptional and posttranscriptional level
* NT reuptake by plasma membrane transporters

Note: NT biosynthetic enzyme and NT
tryptophan hydroxylase -serotonin (=5-HT)
choline acetyltransferase (ChAT) -acetylcholine (Ach)
_glutamic acid decarboxylase (GAD) -GABA

catecholamine NTs: dopamine and noradrenaline
5-HT= 5-hydroxytryptamine

——————-Plasma membrane NT transporters (PMNTTs)
> PMNTTs use transmembrane ion or voltage gradients to energize the reuptake transport. Thus, PMNTTs rely on Na* and K* gradient across the plasma membrane, established by the ubiquitous Na/K ATPase
> PMNTTS are the targets of numerous psychoactive drugs (e.g. cocaine, amphetamine, …) and of drugs used for the treatment of psychiatric disorders, including schizophrenia, depression, and attention-deficit hyperactivity disorder (ADHD).
e.g. cocaine blocks the dopamine PlINTT, amphetamines bind to
PMINTTs responsible for reuptake of NA, DA and 5-HT, with uptake of amphetamine resulting in efflux of these NTs from the cytoplasm to the synaptic cleft; also act on VMAT.
> GABA, glycine, NE, DA and 5-HT transporters belong to a larger family of Na* and Cl-coupled transporters (SLC6 family) with 12 transmembrane domains and cytosolic N- and C-termini
> EAATs belong to a distinct family (SLC1) with 8-10 transmembrane domains

Members of the NE, DA, SERT monoamine transporter family bind extracellularly a Na* ion, the neurotransmitter, and a Cl ion. The transporter then flips to the inside of the cell, where the Na* ion, the neurotransmitter, and a Cl ion are released. After binding of a K* ion, the transporter flips back to the outside of the cell. Thus, the function of the transporter is dependent on the Na/K ATPase.

Question 28

7.6. [vesicular and plasma membrane nt transporters] How is it possible that vesicular neurotransmitter transporters are able to pump neurotransmitters from the cytoplasm (where neurotransmitter concentration is relatively low) into the synaptic vesicles (where neurotransmitter concentration is high)? What fuels this process?

Answer 28

Vesicular neurotransmitter transporters exchange protons for neurotransmitter, and H+ concentration is much higher in the SVs as compared to the cytoplasm (the SV interior is acidic). This H+ electrochemical driving force fuels the import of neurotransmitter into the SVs. This H+ electrochemical gradient is established by the vacuolar-type H+-ATPase, which uses the energy released by ATP hydrolysis to pump protons in the SV lumen.

Question 29

8.1. [postsynaptic organization] how are excitatory and inhibitory postsynaptic densities organized?

Answer 29

reproduce drawing for both

Postsynaptic organization of synapses
> postsynaptic side of the synapse: specialized to receive the NT signal released from the presynaptic terminal and transduce it into electrical and biochemical changes in the postsynaptic cell
> ionotropic NT receptors (=ligand-gated receptor channels) are the cardinal functional components of postsynaptic specializations. These receptor channels are embedded in a dense protein network of:
* anchoring and scaffolding molecules
* signaling enzymes (e.g. kineases, phosphatases)
* cytoskeletal components
* other membrane proteins (Ttranssynaptic / cell-adhesion molecules)
> excitatory and inhibitory postsynaptic specializations differ significantly from each other in terms of molecular organization

Postsynaptic density (PSD) of excitatory synapses
* PSDs are usually located at dendritic spines and contain glutamate receptors AMPA, NMDA)
> Roles of PSDs:
* mediate apposition of pre- and postsynaptic membranes
* cluster postsynaptic receptors
* couple the activation of postsynaptic receptors to biochemical signaling events in the postsynaptic neuron
> PSDs are held together by cytoplasmic actin filaments

Inhibitory synapses are mainly formed on the shaft of dendrites or around the neuronal cell body, and by EM, they show only a slight electron-dense thickening associated with the postsynaptic membrane and hence were described as symmetric (type II) synapses. This presumably reflects the fact that the inhibitory postsynaptic specialization is much less elaborate than the PSD of excitatory synapses.
The cardinal neurotransmitter receptors of central inhibitory synapses are the GABAA receptors and glycine receptors. These receptors interact directly with gephyrin, a well-known postsynaptic scaffold protein of inhibitory synapses

Question 30

8.2. [postsynaptic organization] which classes of molecules are involved? Provide examples for each class.

Answer 30

PSD scaffold proteins and their functional domain
> scaffold proteins bind to and tether or stabilize various membrane proteins and signaling molecules, thereby facilitating coupling of NT receptors with downstream signaling molecules in the PSD
> PDZ Functional domains, typically mediate, protein-protein interactions
Act: actin-binding
Ank: ankyrin repeats
ArfGAP: GTPase-activating protein from Arf small GTPase
CC: coiled coil
CH: calponin homology
CK: calmodulin-dependent kinase (CaMK)-like domain
CRIB: Cdc42/Rac interactive-binding
Efh: EF-hand
EVH1: Ena/VASP homology 1
GH1: GKAP homology
GK: guanylate kinase-like domain
GKBD: GKAP-binding domain
GRKBD: GRK2-binding domain
GTP-CDC: GTP-cell division protein
L27: domain initially found in LIN-2 and LIN-7
LRR: leucine-rich repeat
PH: pleckstrin homology
RapGAP: GTPase-activating protein for Rap small GTPases RasGAP: GTPase-activating protein for Ras small GTPases
RBC: Rac binding
SAM: sterile alpha motif
SHD: Spa2 homology domain
Spectrin: spectrin repeat
ST: subcellular targeting domain
WW: domain with two conserved Trp (W) residues

PSD proteins linked to brain diseases
> ASDs have been linked to mutations in genes encoding Shank2 and Shank3, PSD-93, PSD-95, DLGAP2/SAPAP2, and SynGAP1, as well as synaptic adhesion molecules neuroligin 3, neuroligin 4, and neurexin 1
≥ Mutations in SAPAP3 are associated with obsessive compulsive
*disorder

Question 31

9.1. [synaptic NT-gated receptors] what are the working principles and structure-function of synaptic NT gated receptors?

Answer 31

Synaptic NT-gated receptors
> a.k.a. ionotropic receptors – metabotropic receptors: use second messengers
> decode chemical signals into electrical responses
> ionotropic glutamate, Ach and 5-HT receptors are permeable to cations - > excitation (depolarization) of postsynaptic cell
> GABA and glycine ionotropic receptors are permeable to anions –> hyperpolarization of postsynaptic cell
> Ach, 5-HT, GABA and glycine ionotropic receptors belong to the Cys-loop receptor superfamily –> ionotropic glutamate receptor superfamily

structure of cys loop receptors
> extracellar domains (ECDs) are composed of inner and outer B-sheets and one a-helix per subunit
> transmembrane domains (TMDs) are formed by 4 a-helices/subunit

Cys-loop receptor gene family
> pentameric ligand-gated ion channels with a highly conserved structural signature: a loop formed by a disulfide bridge in the extracellular domain
* nicotinic acetylcholine receptors (nAchR)
* type-3 5-hydroxytryptamine receptors (5HT,R)
* type A and C y-aminobutyric acid receptors (GABAA/cR)
* glycine receptors (GlyR)
> receptor subunit composition generates complexity and diversity, e.g.
* GABAA/cRs: 8 subunit families (a1-6; B1-3; v1-3; 6; €; л; 0; p1-3)
- -> 19 possible subunits!
* nAchR: 5 subunit families (a1-7, 9, 10; B1-4; v; 6; €)
- -> 16 possible subunits; subunit compositions at NMJ:
embryonic: alpha 2 beta gamma ohm ; adult: alpha 2 beta epsilon ohm

Cys-loop receptor gene family: functional aspects
> efficacy of synaptic transmission relies heavily on receptor subunit composition and the number of receptors in the PSD
> receptor-associated molecules provide a scaffold near the surface membrane that influences receptor trafficking through post-translational modification (mainly phosphorylation) of receptors and their associated molecules, receptor expression, and function can be regulated.
> receptor activation is mediated by ligand binding, inducing structural rearrangements around the ligand-binding site which are transmitted through the ECD to the TMDs to initiate ion channel opening.
> ion selectivity filter: at either end of the ion channel, charged rings of residues regulate ion conductance. For nAchRs and5HTyRs, the rings are negatively charged, selecting for cations _, for GABA cRs and GlyRs, the rings are net positively charged and thus anion selective

ionotropic glutamate receptors
> 4 families of iGluR subunits; each subunit is encoded by a distinct gene and there is no mixing between subunit families
> excitatory postsynaptic current is typically mediated by members of the AMPA and NMDA families
> KainateRs are found both on the pre-and postsynaptic compartments and have a more modulatory role
> DeltaRs are incapable to gate an ion channel following ligand binding, and are thus “electrically silent”. Their function is poorly understood.
> Apart from deltaRs, which mainly form homomeric receptors, differential coassembly of subunits generates a large number of iGluR receptor subtypes
> sigmiloR as the cys- loop

Question 32

9.2. [synaptic NT-gated receptors] understand and explain the factors that influence the function of synaptic NT-gated receptors

Answer 32

Modification of iGluRS -, further diversity
> posttranscriptional modifications:
* alternative splicing ~y. ENCODE SINGLE GENED
* RNA editing: leads to single amino acid changes with important functional consequences

> posttranslational modifications:
* phosphorylation: by a variety of intracellular kinases, including
PKA, PKC, CamKIl, Sre and Fyn kinases. Phosphorylation, AGnOVE/ ADD & regulates receptor trafficking, localization, binding to intracellular Proteins and in some cases channel activity
* palmitoylation ( SEE EROLyPOSIS LECarRE
* SUMOylation (SimiLAR TO UBITIFIQUATION)
subunit composition and modification result in unique functional properties of a given receptor
the intracellular tails of iGluR subunits also directly interact with numerous other proteins e.g. scaffolding proteins

Sumoylation is a post-translational modification process. It is analogous to ubiquitylation in terms of the reaction scheme and enzyme classes used, but rather than conjugation by ubiquitin, sumoylation involves addition of SUMOs (small ubiquitin-like modifiers).

> iGluRs form tetramers; each subunit
consists of:
* a large extracellular N-terminal domain (NTD)
* an agonist-binding domain (ABD)
* a transmembrane domain (TMD) that forms the ion channel
* a cytoplasmic C-terminal domain (CTD)

structural mechanisms of iGluR activation (see pic)
> 1 iGluR ABDs display a clamshell-like fold - agonists bind to the interlobe cleft - cleft closure - 2 tension on the ABD-TMD linker - channel gate opening

> competitive antagonists: bind to the ABD, but lock it in an open cleft conformation (doesn’t close cleft but glu can’t bind and receptor channel stays closed)

> partial agonists: induce less domain closure than full agonists, leading to submaximal channel opening

Question 33

9.3. [synaptic NT-gated receptors] Which iGluR families mediate postsynaptic currents and how can their activity be adapted/modulated?

Answer 33

*-iGluR receptor families mediating postsynaptic currents: AMPA and NMDA receptors.
-In general, receptor activity is modulated by the intrinsic activity of the receptors, and by their surface expression levels. How are these modulated?
1. subunit composition: differential co-assembly of subunits generates a large number of iGluR receptor subtypes.
2. posttranscriptional modification of iGluR subunits:
-alternative splicing
-RNA editing, giving rise to single amino acid changes with important functional consequences.
3. posttranslational modification of iGluR subunits:
-phosphorylation: regulates receptor trafficking, localization and binding to intracellular proteins and in some cases receptor activity.
-palmitoylation
-SUMOylation

Question 34

10.1. [electrical synapses] - explain the differences between electrical and chemical synapses

Answer 34

• electrical and chemical synapses may co-exist
• electrical synapses are faster and mostly bidirectional. Some molecules can be found in both chemical and electrical synapses (e.g. caMKII) and others only in chemical (e.g. PSD-95) or electrical (e.g. ZO-1).
• gap junction diameter is larger than in voltage ion channels so it allows the exchange of non-ionic materials (i.e. second messengers, atp, metabolites)
• electrical (ionic) exchange between pre and post synapses
• passive current flow across the gap junction is fast, virtually instantaneous and results in synchronized electrical activity among neural populations

Question 35

10.2. [electrical synapses] - describe and schematically draw the structure of gap junctions

Answer 35

see picture on slides

• gap junctions have pairs of hemichannels (each consists of a hexameric complex of connexions)
• in mammals there are around 20 different connexins. At least five are in the ns (Cx36,45,50,57,30.2)
• Two hemichannels with the same (homotypic) or different (heterotypic) connexin composition can form gap junctions
  NB. possible heteromeric combinations are limited by the restricted temporal and spacial expression pattern of connexins.
  2. While some connexins appear to be promiscuous in their
  interaction partners, others are quite restrictive in those with which they can form partners.
  3. connexins have 4 transmembrane domains

Question 36

10.3. [electrical synapses] - summarize how electrical synapses are complex molecular assemblies, describe the functional classes of molecules involved and provide examples of each class
discuss the diversity of electrical synapses and their distribution in the mammalian CNS

Answer 36

electrical synapses are complex. They’re composed of

-channels: mediate transmission of ions and small molecules
-adhesion complexes: mediate apposition of membranes and
clustering of channels
-scaffolds: mediate clustering of channels
-regulatory proteins: involved in modulation of coupling strength
-trafficking proteins: regulate insertion and removal of channels

On EM images of electrical synapses: electron-dense
material at
each side of the gap junction = electrical synaptic density (ESD)

Example of Cx36 interacting scaffolding protein: zona occludens 1 (ZO-1): interacts with ‘multi-PDZ domain protein 1”
(MUPP1), which in turn interacts with CaMKII (= regulatory protein)

Note: establishment of new gap junction
channels: new connexins are trafficked in
vesicles from the Golgi as undocked
hemichannels. Hemichannels are then
inserted in the plasma membrane at the
periphery of existing gap junction plaques,
rapidly docking with hemichannels
inserted in the apposed membrane

they’re in multiple brain areas, including: retina, olfactory bulb, cortex, hippo, thalamus, hypothalamus, olives, brainstem, spinal cord, gastrointestinal system.
They are also diverse: 1. various neuronal types are coupled with electrical synapses, 2. distinct subcellular sites at which es occur, 3. connexin proteins that form neuronal gap junctions induce diversity, 4. gap junctions occur in a wide variety of ultrastructural configurations

examples:
1. Disparate neuronal types are coupled by electrical synapses
a. inhibitory GABA-ergic interneurons
b. excitatory glutamatergic neurons
c. excitatory cholinergic neurons
d. excitatory peptidergic neurons
e. excitaton) noradrenergic neurons
2. Distinct subcellular sites at which electrical synapses occur
a. purely electrical dendro-dendritic synapses
b. purely electrical somato-somatic synapses
c. axo-axonic electrical synapses
d. excitatory mixed synapses: combined chemical plus electrical synapses; axo-somatic or axo-dendritic
e. “reciprocal” (mirror) dendro-dendritic mixed synapses
3. the connexin proteins that form neuronal gap junctions induce diversity
4. gap junctions occur in a wide variety of ultrastructural configurations

Question 37

10.4. [electrical synapses] - paraphrase the mechanisms that underlie plasticity of electrical synapses, as well as their time scales and signaling pathways involved; predict the effect of a given manipulation on coupling strength.

Answer 37

see figures!!
Mechanisms that alter coupling on three time scales:
1. Short-term change in coupling (B): resulting from activation of R2 postsynaptic receptors on one of the two coupled cells » drop in R2 + drop in coupling coefficient.
time scale: milliseconds, may last a few seconds
2. Intermediate term changes in Rz coupling (C): resulting from modification of existing gap junction channels, such as phosphorylation (reduces Ri) or dephosphorylation (increases Rj).
time scale: seconds to minutes; may last for hours.
3. long-term changes in coupling (D): resulting from decrease or increase in the number of gap junction channels due to expression or turnoverchanges.
time scale: minutes to days.

Notes: 1. the type of connexin that constitutes the gap junction has dramatic effects on channel conductance 2. it is thought that only a small fraction (<20%) of the channels in an electrical synapse are functional and efficient to support electrical transmission, suggesting that a heterogeneous population of channels can coexist in a gap junction plaque. Obviously, if a higher fraction of channels is active, junctional resistance will go down and coupling efficiency will increase.

mechanisms controlling plasticity of photoreceptor electrical synapses
Coupling between mammalian photoreceptors: high at night (darkness), low in the daytime (light).

‘Mechanism 1: daylight 3 increased dopamine secretion » D4 receptor activation on photorecpetors 9 inhibition of adenylate cyclase through Gi signaling - reduced cAMP production
3 reduced activity of cAMP-dependent protein kinase (PKA) - reduced Cx36 phosphorylation - reduced photoreceptor coupling.
note: intermediate term plasticity is controlled by a balance btw protein kinase and phosphatase activities that control phosphorylation state of the connexions

Mechanism 2: adenosine controls photoreceptor coupling through both adenosine A2a and A1 receptors. Extracellular adenosine levels: highest at night (darkness) - adenosine A2a receptor activation - adenylate cyclase activation through
Gs signaling - increased PKA activity - increased
Cx36 phosphorylation.
The A1 receptor has higher affinity for adenosine than A2a - A1 is active during daytime » inhibition of adenylate cyclase through Gi signaling »
reduced PKA activity reduced Cx36 phosphorylation
Note: a second phosphatase, PP1, appears
to target PP2A to put the brakes on the
pathway
amacrine cells: inhibitory interneurons in
the retina; AII amacrine cells use glycine
as neurotransmitter; they capture cellular
input from rod bipolar cells and
redistribute it to cone bipolar cells

Question 38

10.5. [electrical synapses] - explain the methods used to study electrical synapses and be able to select the right method to tackle a given scientific problem

Answer 38

1. Freeze-fracture replica immunogold labeling (FRIL):
   -rapidly frozen biological samples are fractured, ‘replicated’, immunostained with gold particle-labeled antibodies and analyzed by transmission electron microscopy (TEM).
   -allows for high-resolution, semi-quantitative analysis of symmetric versus asymmetric connexin labeling in matching apposed hemiplaques of individual ultrastructurally visualized gap junctions.
   Notes: ’Matched-double-replica FRIL; larger dots in image: Cx36 labeling; smaller dots: Cx45 labeling
   *fracturing (= cracking) of frozen tissue is done using a microtome, a knife-like instrument for cutting thin tissue sections. This fracture occurs along weak portions
   of the tissue such as membranes or surfaces of organelles. Following fracturing, the sample undergoes a vacuum procedure, called “freeze etching.” The surface of the fractured sample is shadowed with carbon and platinum vapor to make a stable replica, which follows the contours of
   the fracture plane.
2. Dual cell recording under direct visualization:
   -electrophysiological analysis involving simultaneous recordings of neighboring cells using patch-clamp electrodes, with the aim to detect electrical coupling.
   -this can be combined with the use of transgenic mice expressing EGFP in select neuronal populations, e.g. parvalbumin expressing cells or Cx36 expressing cells
3. Intracellular dye- or tracer injection and subsequent visualization of tracer transferred
   between cells via gap juctions:
   -allows for analysis of neuronal gap junction coupling
   -examples of tracers: neurobiotin, biocytin
4. Gap junction blockers:
   e.g. mefloquine has high specificity for Cx36-containing gap junctions
5. Electrophysiological analysis of ‘spikelets’ or ‘fast pre-potentials’:
   Transmission of spikes (action potentials) through electrical synapses evokes corresponding coupling potentials in a postsynaptic cells, roughly resembling the tire course of the presynaptic action potential.
   These so called ‘spikelets’ or ‘fast pre-potentials’ can be spontaneously observed in electrically-coupled networks

Question 39

10.6. [electrical synapses] _ explain the potential functions of electrical synapses and their links to disease, and deduce the functional consequences of electrical synapses in a given neuronal network

Answer 39

functions of electrical synapses
1. Promotion of synchronous activity:
Transmission at electrical synapses is usually bidirectional, and therefore changes in membrane potential of one cell within an electrically-coupled network are presumably shared with all the partners in the network.
This allows for high-speed neuronal network oscillatory activities, which are thought to be involved in e.g. learning and memory and consciousness.

2. Signal amplification:
   e.g. ‘lateral amplification’ increases the sensitivity of sensory neuronal networks.
   Figure panel: mechanosensory activation of the crayfish lateral giant neurons that command the tailflip escape response
   mechansensory
3. Noise reduction
   e.g. photoreceptors in the vertebrate retina: their electrical coupling promotes a decrease of uncorrelated noise and a relative amplification of correlated visual signals

link to disease: A mutation in a non-coding (regulatory) region of the C×36 gene has been linked to juvenile epilepsy

Question 40

11.1. [nmj] explain and schematically draw the structural organization of the NMJ (cellular and molecular)

Answer 40

> Neuromuscular junction (NMJ):
* synapse between motor neurons and skeletal muscle fibers
* allows posture, movement and respiration
* NMJ dysfunction/degeneration occurs in several neuromuscular diseases, e.g. myasthenia gravis, ALS, SMA,

• Neurotransmitter: ACh
  Postsynaptic receptor: nicotinic ACh receptor (nAChR)
• member of the Cys-loop receptor family
• nAchR: 5 subunit families (a1-7, 9, 10; B1-4; gama; ohm; epsilon); subunit compositions at NMJ:
  embryonic: a2By ohm; adult: a2b epsilon ohm

Organization of NMJ
A: motor neurons whose soma and dendrites are located in the spinal cord send their
axons to the periphery and form neuromuscular junctions to innervate skeletal
muscle fibers. Axons are wrapped by myelin sheaths formed by Schwann cells. At the
site of neuromuscular contact, axons ramify into branches and form presynaptic
nerve terminals that are capped by terminal Schwann cells and covered by
kranocytes. Note the accumulation of fundamental myonuclei (black) and
postsynaptic folds at the site of the nerve-muscle contact.
B: high magnification of the neuromuscular junction. In addition to ACh-filled vesicles,
local specializations in the presynaptic motor nerve terminal include active zones
(where vesicles fuse with the terminal membrane) and a high number of
mitochondria (brown). Postsynaptic specializations in the skeletal muscle fibers
include folds that form opposite the active zones, aggregates of AChRs (red) at the
crest, and high concentrations of NaV1.4 (green) in the troughs of the folds. The
localization and high concentration of AChR and NaV1.4 are important for efficient
neuromuscular transmission.
C: whole mount view of the synaptic band of a mouse diaphragm. Motor axons are
labeled with antibodies to growth-associated protein 43 (GAP-43; green), and
postsynaptic AChRs are visualized by BTX (red).

Question 41

11.2. [nmj] describe the different stages of NMJ development, including the cellular and molecular interactions involved

Answer 41

(A) At early embryonic stages (E11 to E13.5 in mice), primary myotubes are formed
before innervation by the motor nerve
(Aa) A central zone of AChR clusters without contact to motor axons is formed (a
process called ‘pre-patterning’). Some myonuclei in the center of the muscle
express higher levels of Musk than those in the periphery.
(Ab) At the beginning of motor innervation of the primary myotubes, the number of
myonuclei with high Musk levels increases, and myoblasts proliferate in the
center inside the basal laminae of the primary myotubes.
(Ba) During late embryonic development (E14.5 to postnatal stages), muscle size
increases due to formation of secondary myotubes through fusion of proliferating
myoblasts.
In primary myotubes, innervation initiates transcription of synaptic genes in
myonuclei underlying the neuromuscular contact. The nuclei are called
‘fundamental’ or ‘subsynaptic’ nuclei. AChR cluster size, number and shape are
refined with innervation (E14) that disperses non-synaptic AChR clusters and
induces new neural AChR synaptic clusters through the secretion of Agrin.
Initially, the secondary myotubes are electrically coupled to the primary myotubes
through gap junctions.
(Bb) Subsequently, secondary myotubes segregate from the primary myotubes and
secrete their own basal lamina. At this stage, myotubes are multiply innervated,
and electrical activity leads to condensation of AChRs to the site of innervation.
Furthermore, expression of synaptic genes is suppressed in nonsynaptic nuclei and selectively stimulated in subsynaptic nuclei.
(C) In mature muscle, each fiber is innervated by one motor neuron. Excessive
innervation is removed during the process of synapse elimination.

Question 42

11.3. [nmj] explain and schematically draw the molecular mechanism underlying selective expression of AChR subunit genes in subsynaptic nuclei

Answer 42

-Agrin is a proteoglycan synthesized by the MN and the muscle cell, but only the
nerve-secreted isoform is active and induces AChR clustering. Agrin binds LRP4 but its
post-synaptic effect is transduced through MuSK activation.
-Dok-7 is a muscle-specific adapter protein that is recruited by MuSK once
phosphorylated and induces a cascade of phosphorylation that leads to AChR
clustering.
-In adult/mature NMJs, action potential activity suppresses synaptic genes in
nonsynaptic myonuclei, while their expression is maintained in the fundamental nuclei
by agrin-Lrp4-MuSK signaling and the subsequent activation of transcription factor
Erm, which drives expression of AChR-encoding genes (Chrn) and Musk.

Question 43

11.4. [nmj] describe the cellular mechanisms that regulate AChR surface expression levels

Answer 43

Parallel signaling pathways at the NMJ are involved in the coordinated maturation of
the presynaptic terminal (green), the postsynaptic muscle fibre (red) and the
perisynaptic Schwann cells (PSCs; grey); dotted lines indicate pathways that are still
being debated. Agrin, which is released by the nerve terminal, muscle fibre and
surrounding PSCs, acts on the LRP4–MUSK complex, which comprises low-density
lipoprotein receptor-related protein 4 (LRP4) and muscle-specific tyrosine kinase
receptor (MUSK). The phosphorylation of MUSK leads to rapsyn-mediated clustering
of ionotropic acetylcholine receptors (AChRs) and postsynaptic maturation. AChR
clustering can also be enhanced by WNT ligands (associated with MUSK in the figure),
whereas release of ACh inhibits AChR clustering. LRP4 is mainly of postsynaptic origin,
although neuronal LRP4 may also have a role. LRP4 acts as a co-receptor for agrin and
stimulates AChR clustering as well as presynaptic maturation by clustering synaptic
vesicles and active-zone proteins. Neuregulin 1 (NRG1) can be released by the nerve
terminal and/or surrounding PSCs, and binds to PSC-expressed or postsynaptic ERBB
receptors (ERBB2 or ERBB3). The binding of NRG1 to ERBB receptors on PSCs
promotes PSC survival and maturation. Although the exact signaling pathway is
controversial, the activation of postsynaptic ERBB receptors by NRG1 may increase
levels of postsynaptic proteins such as rapsyn, MUSK and AChR, and could have a role
in AChR clustering, which overall leads to postsynaptic maturation. Synaptic laminins,
such as laminins β2, α4 and α5, are released by the muscle fibre. They form
heterotrimeric glycoproteins that are included in the basal lamina and are important
for proper pre- and postsynaptic alignment and maturation, as well as PSC maturation (not shown). Laminin β2 binds to presynaptic calcium channels and regulates activezone proteins (not shown). Postsynaptically, laminins interact with integrin β1, which
increases AChR clustering. Fibroblast growth factors (specifically FGF7, FGF10 and
FGF22) are released by the muscle fibre and activate mainly presynaptic type 2B FGF
receptors (FGFR2B); thus, they are important for vesicle clustering and presynaptic
maturation, as are type IV collagen α chains (α2, α3 and α6). PSC-derived
transforming growth factor-β (TGFβ) induces presynaptic maturation and
postsynaptic differentiation by upregulating the expression of agrin. Synaptically
released ATP is detected by PSC-expressed purinergic type 2Y receptors (P2YRs) and
triggers increases in intra-PSC Ca2+ concentrations. PSCs also express muscarinic
AChRs (mAChRs), and their activation by the local application of ACh triggers
increases in intra-PSC Ca2+ concentrations. However, mAChRs are not activated by
endogenous ACh release. Matrix metalloproteinases (MMPs) in the extracellular
matrix (ECM) that surrounds PSCs regulate the composition of the ECM and cleave
matrix proteins such as agrin, triggering its removal from the ECM.

structural components involved in NMJ function
AchE is localized to the synaptic basal lamina and is essential to inactivate ACh. The
homotetrameric subunits co-assemble with the triple helical collagen tail, termed
ColQ, which tethers the entire enzyme to the synaptic basal lamina.
The dystrophin glycoprotein complex (DGC) contains dystroglycan, which is
posttranslationally cleaved into α-dystroglycan (αDG) and the transmembrane
component β-dystroglycan (βDG), the sarcoglycans (α through δ) and sarcospan (ss).
βDG associates with rapsyn to link AchRs to the DGC and connects to α-dystrobrevin
(αDB), to α-syntrophin (syn) and utrophin. Utrophin links the entire complex to the Factin cytoskeleton.
The voltage-gated sodium channels (Nav 1.4) are localized to the throughs of the
synaptic cleft.
Note: Rapsyn (receptor associated protein of the synapse) is a membrane-bound
cytoplasmic molecule that binds AChR and is necessary for AChR clustering

dynamic of the AchR at NMJ
Nicotinic acetylcholine receptors (nAChR) subunits are synthesized in the ER and
exported to the muscle plasma membrane. From the ER, instead of being targeted to
the cell surface, most nAChR subunits are degraded by the ER-associated ubiquitinproteasome degradation pathway. In the postsynaptic membrane, there is significant
lateral diffusion between the synaptic and perisynaptic membrane spaces. Lateral
diffusion of nAChRs from the perisynapse into the NMJ contributes significantly to
maintain the synaptic receptor density. Conversely, when receptors escape from the
postsynaptic density into the perisynaptic space, there is significant internalization of
nAChRs into endosomal compartments. Trafficking through the endosomal pathway,
a fraction of internalized nAChRs is targeted for degradation. However, a significant
portion of those nAChRs actually recycle back into the synaptic membrane,
contributing to the maintenance of the synaptic nAChR pool. Most of these dynamics
are tightly regulated in the NMJ by several stimuli, such as synaptic activity or
association of dystrophin glycoprotein complex components.

Question 44

11.5. [nmj] discuss the functional properties different muscle fiber types and their differential vulnerability in neuromuscular disease

Answer 44

muscle metabolism
Scheme showing some differences in glucose, lactate, and fatty acid metabolism
between fast and slow muscle fibers. Pathways prevalent in fast or slow muscle fibers
are shown as red or green arrows, respectively. DHAP, dihydroxyacetone phosphate;
GLUT4, glucose transporter 4; F-6-P, fructose-6-phosphate; FAT/CD36, fatty acid
translocase; FFA, free fatty acids; F-1,6-P, fructose-1,6-bisphosphate; F-2,6-P, fructose-
2,6-bisphosphate; G-3-P, glyceraldehyde-3-phosphate; G-6-P, glucose-6-phosphate;
GPD1, glycerolphosphate dehydrogenase 1 (cytoplasmic); GPD2, glycerolphosphate
dehydrogenase 2 (mitochondrial); HK, hexokinase; LDH, lactate dehydrogenase;
MCT1, monocarboxilic acid transporter 1; MCT4, monocarboxilic acid transporter 4;
PDH, pyruvate dehydrogenase; PFK, phosphofructokinase; PFKFB3,
phosphofructokinasefructose bisphosphatase 3; TG, triglycerides.

Muscle fiber type-selective vulnerability of motor axons in ALS

In SOD1-ALS mouse models:
-axons of fast-fatigable MNS:
degenerate presymtomatically
-axons of fast fatigue-resistant
MNs. degenerate at symptom onset
-axons of slow MNs: resistant to degeneration

Question 45

12.1. [als] describe the symptoms of ALS and explain how they arise

Answer 45

> adult-onset neurodegenerative disease
characterized by selective and progressive degeneration of motor
neurons

lower motor neuron degeneration
* muscle weakness
* muscle atrophy
* fasciculations
upper motor neuron degeneration
* spasticity
* slowness of movement
* incoordination

muscle atrophy= shrinkage of muscle, muscle wasting.
fasciculations= twitching = a brief spontaneous contraction affecting a small number
of muscle fibers, often causing a flicker of movement under the skin. spasticity= involuntary tightness of a muscle, some muscles are continuously contracted.

Question 46

12.2. [als] summarize the genetics of ALS, and understand how ALS animal models can be generated based on familial ALS genes

Answer 46

.

Question 47

12.3. [als] Paraphrase and design experimental approaches to evaluate whether non-cell-autonomous toxicity contributes to motor neuron degeneration in ALS

Answer 47

.

Question 48

12.4. [als] explain how loss-of-function changes can be distinguished from gain-of-toxic-function changes in ALS animal models

Answer 48

.

Question 49

12.5. [als] design experimental approaches to investigate the contribution of skeletal muscle pathology in ALS mouse models

Answer 49

.

Question 50

13.1. [practical]
Question 1 (Research strategy – open question that gave you the opportunity to speculate on the topic): give a short research strategy (step-wise protocol) how you will acquire a molecular signature of idiopathic ASD using iPSC-derived brain organoids. What are the steps involved?

Answer 50

(1) Collection and preparation of material; fibroblast-derived iPSCs obtained from skin biopsies of four families (idiopathic ASD proband with increased head circumference; male individuals; parents not affected: likely de novo mutation) and four unaffected, first-degree male family members (Figure 1) were differentiated into organoids (Figure 3).

Question 51

13.2. [practical] Question 2 (Approach):
(2) Explore molecular ASD organoid signatures at the DNA-, RNA- or protein level?

Answer 51

Approach at the DNA level: single-nucleotide variation (SNV) or copy number variation (CNV) microarrays; whole-genome (exome) DNA sequencing
Approach at the RNA level: mRNA expression profiling by microarrays or RNA-sequencing (RNA-seq)
Approach at the protein level: (phospho)proteomics (mass spectrometry)

Approach at the DNA level:
Identify a (deleterious) genomic variation contributing to ASD: whole-genome sequencing of fibroblasts/iPSC genomic DNAs for SNV and CNV discovery: no obvious genomic alterations (no do novo rare SNVs and/or CNVs);
Why genomic DNAs from fibroblasts, iPSCs and organoids?: any experimental manipulation (reprogramming; in vitro culturing) may introduce additional genomic variants not linked to ASD; so best choice (close to in vivo): fibroblasts.

(b) Approach at the RNA level: mRNA expression profiling by microarrays or RNA-seq (has become our choice).

(c) Approach at the (phospho)protein level: proteomics, and in particular phosphoproteomics, is not yet fully developed on a genome-wide scale (i.e. much less developed than those at the DNA- and RNA-levels) and clearly more difficult than DNA/RNA work: also: proteins display post-translational modifications; still: proteins are directly relevant for identifying and validating the molecular pathway/signature involved.

Question 52

13.3. [practical] Question 3 (Brain region related to the generated organoid; regional specification):

Answer 52

Approach: RNA-seq of control (better than ASD) iPSC-derived organoid and compare with database transcriptomes of human brain regions (BrainSpan atlas of 16 male/female pre- and postnatal (sub)cortical human brain structures).
Result: Organoid transcriptome most similar to human dorsal telencephalon (cerebral cortex and hippocampus) transcriptomes (Figure 4).

RNA-seq advantages over microarray:
More (new?) transcript sequences identified (whole transcriptome; noncoding RNAs)
Genetic variations identified
More quantitative

Transcriptome analysis
Approach: RNA-seq of iPSC-derived organoids from four ASD/four respective fathers.
Result: Terminal differentiation day (TD) 11: 1,062 differentially expressed genes (DEGs);
TD31: 2,203 DEGs.

Question 53

13.4. [practical] first next step

Answer 53

Validation by quantitative polymerase chain reaction (qPCR), a relatively easy, cheap and fast method. Selection of RNAs/genes to be validated: randomly select a number (10 or so) from the list of differentially expressed RNAs/genes; use same RNA samples for qPCR- as used for RNA-seq-analysis. Result:0.98 correlation coefficient; 100% concordance in direction of change (Table 1).

Question 54

13.5. [practical] Question 5 (in Table 1, DLX1/2; GAD1/2; Vglut1/2: small gene families; specific mRNA quantification by qPCR: primer pairs directed towards the 3’-untranslated regions (3’ –UTRs). Why?):

Answer 54

Hint: members of a gene family are structurally related (at both the nucleotide and amino-acid sequence level).
Answer: gene family members are structurally related, especially in the protein-coding regions, so it is safer to design the primer pairs to a region in the mRNAs that in general is less conserved, i.e. the 3’ –UTR; exceptions to choose from in the 3’-UTR are miRNA-binding sites (they are in general well conserved).

PCR:
Traditional or Reverse Transcriptase (RT-PCR) method: final phase or end-point product detection on agarose gel and quantified by densitometric scanning
Quantitative (q) PCR

Question 55

13.6. [practical] Question 6:
Potential problem with quantification by RT-PCR? How circumvented with qPCR?

Answer 55

.Potential problem with quantification by RT-PCR?
Answer: Plateau in the amount of PCR-product (already at 20 or 25 cycles); gives only a semi-quantitative
estimate; partial solution: use various cycle numbers or various cDNA dilutions.

How circumvented with qPCR?
Answer:
Real-time qPCR (monitor the progress of the PCR as it occurs, i.e., in real time): continuously determines the amount of PCR-product (collect data while the reaction is proceeding). Measurement is at the exponential phase of the PCR reaction (i.e., the optimal point for analyzing data).
Gene co-expression network analysis:
24 modules of coexpressed genes (corresponding to about 17,298 genes) across ASDs and controls at TD11 and TD31 determined with network analysis software program(s), e.g. WGCNA (Weighted Gene Co-expression Network Analysis)
Modules’ eigengenes (i.e., the first principal component of theexpression profiles of themodule’s genes, representing the module by an expression value) and assess their changes over time (TD11; TD31) and across diagnosis (Figure 6)

Enriched in upregulated genes (Figure 7):
Blue module: ‘neuronal differentiation’ (Gene Ontology, GO, term)
Magenta module: ‘regulation of transcription’

Enriched in upregulated genes only at TD31:
Brown module: ‘synaptic transmission’

Functionally validate blue and brown modules
Approach: morphometric cellular analyses and immunostaining for the markers
MAP2 (microtubule-associated protein 2) for neuronal differentiation
VGAT (vesicular GABA transporter) for inhibitory neurons
VGLUT1 (vesicular glutamate transpoter-1) for excitatory neurons (Figure 8

Question 56

13.7. [practical] Question 7 (Conclusion from Figure 8?):

Answer 56

Answer - In ASD-derived neurons:
MAP2: significant increase in neuronal maturation; in agreement with the upregulated expression of the blue module, accelerated or increased neuronal differentiation in ASD
VGAT: significant increase in inhibitory synapses
VGLUT1: no significant changes in excitatory synapses

Bias for differentiation into specific neuronal subtypes?
Approach: use telencephalic developmental transcription factors (TFs; from blue and magenta modules) as markers (Figure 9).
Result:
TBR1 (cortical excitatory neuron precursors of layer 6 neurons): not significantly different
CTIP2 (early-born layer 5 neurons): not significantly different in ASD

DLX1-2 (GABAergic inhibitory neuronal fate marker): increased significantly in ASD
GAD1/GAD67 (GABA-synthesizing enzyme): increased significantly in ASD organoids compared to those from unaffected family members

Bias for differentiation into specific neuronal subtypes?
Approach: use telencephalic developmental transcription factors (TFs; from blue and magenta modules) as markers (Figure 9).
Result:
TBR1 (cortical excitatory neuron precursors of layer 6 neurons): not significantly different
CTIP2 (early-born layer 5 neurons): not significantly different in ASD

DLX1-2 (GABAergic inhibitory neuronal fate marker): increased significantly in ASD
GAD1/GAD67 (GABA-synthesizing enzyme): increased significantly in ASD organoids compared to those from unaffected family members

Question 57

13.8. [practical] Question 8 (Quantification of GAD1 and GAD2 proteins on Western blot (Figure 10)?):

Answer 57

Answer:
Densitometric scanning of the protein bands
Necessary: quantification relative to the bands of the housekeeping protein GAPDH

Result:
Increased protein expression of the GABAergic inhibitory interneuron marker proteins GAD1/2

Together, the cellular analyses strongly suggest an overproduction of progenitors and neurons of the GABAergic lineage as well as an altered balance between the number of excitatory and inhibitory neurons in ASD organoids (Figure 9, below, middle figure, framed

Refinement of our understanding of the functional annotations of the upregulated magenta ‘neuronal’ module.

Approach:
Investigate its (transcription-related) canonical pathway annotation
overall: magenta-module genes function in the transcriptional regulation of cell fate and cell proliferation in the forebrain
Select one of the significantly upregulated TF genes in the magenta module: TF crucial for acquisition of neural cell fates and precursor cell proliferation in the telencephalon

Question 58

13.9. [practical] Question 9 (Which considerations/criteria do you propose for selection of the most attractive hub gene from Figure 11/12? Important decision – follow-up is time consuming):

Answer 58

Answer - FOXG1 (arrow in Figure 12):
Would be good to refine the network by extensive literature analyses as a first step in the selection of the key gene (key target) in the network/pathway; compound modulating such a key target could (eventually) result in autism drug
One of the top 100 magenta hub genes
Consistently among the top 10 upregulated genes (8.5-/13-fold expression increase at TD11 and TD31, resp.)
TF important for telencephalon development
Knock-in/-out cell/animal models available?
Loss-of-function FOXG1 mutations occur in atypical Rett syndrome patients (an ASD-related disorder) and confer a small brain size; ASDs studied here have large brain size: FOXG1 may be, at least in part, involved in brain size and social disability phenotypes

Question 59

13.10. [practical] Question 10 (Approach to test the hypothesis that abnormally high levels of FOXG1 and its downstream genes could be responsible for the phenotypic abnormalities identified in neuronal cells of macrocephalic ASD patients?):

Answer 59

Answer:
Generate four stable iPSC lines from ASD-derived iPSC line 07-P#9 to downregulate FOXG1 (using lentiviruses carrying short hairpin RNAs – shRNAs – specifically targeting FOXG1 or a non-targeting negative control shRNA consisting of a scrambled oligonucleotide sequence)
Test whether some of the neurobiological alterations are reverted

Question 60

13.11. [practical] Question 11:
How to check the degree of downregulation by shRNA (at the RNA as well as the protein level)?

Answer 60

Answer:
qPCR analyses at TD11 (Figure 13): two FOXG1-targeting shRNAs (shRNA-2 and 3) downregulate FOXG1 mRNA expression; FOXG1-targeting shRNA-1 does not result in FOXG1 downregulation
immunostaining for FOXG1 (Figure 14): shRNA-2 and -3 downregulate also at the protein level

Why don’t we use just one but rather three shRNAs targeting FOXG1?
Answer:
The effectiveness of mRNA downregulation by shRNAs is difficult to predict: use three different shRNAs, each directed towards a specific region within FOXG1 mRNA (in general, one needs at least two working shRNAs for a proper downregulation study)

Question 61

13.12. [practical] Question 12 (Approach to prove that FOXG1 causes the overproduction of GABAergic interneurons observed
In the ASD-derived organoids?):

Answer 61

Answer:
AnalyzeGABAergic marker expression following FOXG1 downregulation
qPCR: downregulation of GABAergic markers DLX1, DLX2, and GAD1, but not the dorsal forebrain marker PAX6 (Figure 15)
Immunostaining: The normal levels of GABAergic neuronal differentiation (DLX1-2- and GAD1-positive cells) are restored (Figure 16; shRNA-3; overproduction with shRNA-C)
No or minor effects on the transcript/protein expression levels of dorsal forebrain markers (such as PAX6), or on TFs directing cortical excitatory neuron differentiation (such as TBR1) (Figures 15 and 16)

Thus:
FOXG1 is involved, at least in part, in causing the overproduction of neurons of the GABAergic lineage found in ASD proband-derived organoids
The early increase in proliferation of GABAergic neuronal progenitor cells gives rise to an increased proportion of mature GABAergic interneurons
FOXG1 RNAi restores both these early and late effects to levels comparable to those found in unaffected family members
Upregulated FOXG1 expression in ASD neural cells is driving an early proliferative effect in neuronal precursor cells of the GABAergic lineage
FOXGI is part of the molecular signature of idiopathic ASD

Highlights
iPSC-derived telencephalic organoids reflect human midfetal telencephalic development
Inhibitory neurons are overproduced in organoids from patients with idiopathic ASD
Overproduction of inhibitory neurons is caused by increased FOXG1 gene expression

Question 62

13.13. [practical] Question 13 (Major overall flaw/shortcoming of our study?):

Answer 62

Hint: Type of differentiated iPSCs used to obtain brain organoids and the complexity of the brain.

Answer:
Organoids used here: radial glia, intermediate progenitors and neurons; the length of the time of differentiation was relatively short (maximum of 31 days of terminal differentiation, TD31), which limits the diversity of brain cell types generated
Brain: more cell types (e.g. oligodendrocytes, microglia and astrocytes; not co-developing alongside the neuronal ones and are therefore not represented in the organoids)
Thus: organoid recapitulates only a subset of brain components and thus of cell-to-cell signaling processes

Question 63

13.14. [practical] conclusions?

Answer 63

iPSC-derived cortical organoids recapitulating human telencephalic development
Genome-wide transcriptome analysis in four families affected by idiopathic ASD
Affected individuals do not share any obvious underlying genomic alterations (heterogeneity in genotypes)
All individuals express a phenotypic trait that confers increased symptom severity, namely macrocephaly
Identification of perturbations in coherent programs of mRNA expression: upregulation of cell proliferation, unbalanced GABAergic inhibitory neuron differentiation, exuberant synaptic development and a generalized increase in proliferative potential
In accord with earlier hypotheses stating that abnormal control of cell proliferation, overproduction of neurons, increased spine densities, and an imbalance between glutamate and GABA neurons might contribute to the ASD phenotype, including its accelerated brain growth
A shared pathophysiological mechanism may exist for idiopathic ASD
Dysregulated gene expression in iPSC-derived organoids, and FOXGI in particular, could be used as a potential biomarker of severe ASD
Deletions and missense mutations in the FOXGI gene have been associated with an atypical Rett syndrome (a rare form of autism that affects only girls) and small brain size, suggesting that deviations in its expression levels during brain development, in excess and defects, cause opposite modulation in brain growth but a similarly disabling outcome, characterized by intellectual disability and ASD-like symptoms
Directly studying neurodevelopmental processes in patents with neuropsychiatric disorders that have heterogeneous etiologies can open inroads into diagnosis and therapy