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neuroscience > advanced molecules, membranes and cells > Flashcards

advanced molecules, membranes and cells Flashcards

1
Q

Embryonic stem cells are…

A

pluripotent stem cells derived from the inner cell mass of the blastocyst of preimplantation embryos, at which time they consist of 50-150 cells

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2
Q

Entire embryo will derive from the

A

‘inner cell mass’ (ICM)

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3
Q

ICM expresses what marker

A

oct 4 and nanog

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4
Q

dissociating a blastocyst and culturing the ICM produces

A

embryonic stem cells

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5
Q

embryonic stem cells introduced into adult mice will form

A

teratomas

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6
Q

if embryonic stem cells are reintroduced into a blastocyst they will contribute to and form

A

all parts of the embryo forming chimeric mice with ES cells contributing randomly

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7
Q

Oct4 is

A

A transcription factor, expressed in ICM cells

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8
Q

Oct4-null embryos develop to

A

blastocyst stage but die because the ICM cells are not pluripotent

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9
Q

Nanog null ICM cells lose

A

pluripotency and develop as extra-embryonic tissues (parietal endoderm)

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10
Q

Leukemia Inhibitory Factor (LIF) role in ES cell manipulation

A

prevents differentiation into mesoderm/endoderm

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11
Q

BMP role in ES cell manipulation

A

prevents differentiation into neuroectoderm

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12
Q

the important highlights of the maintenance of mouse ES cells pathway

A

Leukemia Inhibitory Factor promotes GP130/LIFR -beta to work on three pathways JAK, Grb2, PI3K which through kinases goes on to influence SOX2 and Nanog which then alter OCT4

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13
Q

how may ELECTROPORATION introduce DNA into ES cells

A

Electroporation blows tiny lesions in cell membranes ⟶ DNA enters before or during repair ⟶ somehow makes its way to nucleus.

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14
Q

process of using ES cells to produce GM mice

A

DNA electroporation->selection->analyse survivors-> inject into ICM of host blastocyst->implant into pseudo pregnant female

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15
Q

some examples of spontaneous mouse mutations

A

small eye mouse
looptail mouse
clubfoot mouse

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16
Q

random mutagenesis involves

A

male animal exposed to mutagens such as ethyl nitrosourea (ENU) or ethyl methansuplhonate (EMS)

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17
Q

ethyl nitrosourea (ENU) mutagenesis induces

A

point mutations by ethylating DNA base pairs in replicating sperm cells

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18
Q

ethyl methansuplhonate (EMS) mutagenesis induces

A

turns G/C base pairs into A/T

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19
Q

low doses of EMS/ENU mutagenesis ensure

A

randomly distributed point mutations at low frequency throughout genome

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20
Q

the point of a mutagenesis screen is to

A

screen for recessive mutations and breed to produce heterozygous mutations

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21
Q

mutagenesis screen benefit

A

Can generate mutations in tissues without a priori assumptions or knowledge about which genes are important

Can generate new alleles of genes that you would never have been able to make deliberately, or would never have thought of

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22
Q

mutagenesis screen limitations

A

Uses very large numbers of animals

Wasteful - you only find what you’re looking for
e.g. limb defects, and may miss interesting mutations just by not looking at the right bit of the animal

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23
Q

Gene Targeting (Knock-Outs) mean

A

To delete genes (‘knock genes out’) from animals by Homologous Recombination in Embryonic Stem Cells

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24
Q

Homologous Recombination Usually occurs during

A

meiosis

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25
Q

Homologous Recombination entails

A

Identical (homologous) sequences on maternal + paternal chromosomes find each other, line up and may ‘cross over

It can occur ‘accidentally’ between identical DNA sequences in any cell at any time ⟶ at low frequency

Can be used to introduce new DNA into cells

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26
Q

in practice genetic manipulation using homologous recombination involves

A

transgene

flanking DNA identical to sequence and hope they cross over

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27
Q

ganciclovir for Homologous Recombination ensures

A

cells with TK are killed (herpes simplex thymidine kinase gene)

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28
Q

where is TK attached on the transgene

A

outside the flanking sequences so it should be lost after undergoing homologous recombination

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29
Q

neomycin for Homologous Recombination ensures

A

cells without neoR are killed

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30
Q

NeoR is attached where on the transgene

A

next to the gene so it should be present after homologous recombination

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31
Q

after positive and negative selection following Integration of Target Vector by Homologous Recombination what should be conducted?

A

PCR and Southern Blotting

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32
Q

following successful targeted ES cells what happens next?

A

inject them into inner cell mass (ICM) of host blastocysts, implant into uterus of pseudopregnant female

Get chimeric offspring that can be used for breeding

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33
Q

Homologous Recombination accounts for what percentage of all integration events

A

<2%

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34
Q

issues with Knockouts

A

Redundancy can lead to mild or no phenotype (at the level of analysis)

Early embryonic lethality may prevent analysis of later events

Genetic background
The strain of mouse you use may affect the phenotype you see

Genes don’t act in isolation, and there may be many steps between gene dysfunction and animal phenotype

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35
Q

Eukaryotic genes need:

A

1) A promoter that will drive expression in the tissues you require…
2) An open reading frame encoding the gene you want to express…
3) Sequences that ensure correct mRNA processing

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36
Q

what signal ensures correct mRNA processing?

A

polyadenylation signal

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37
Q

cDNA refers to

A

complementary DNA - Contains coding region of gene, with no introns

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38
Q

Minimal promoter is where

A

transcriptional machinery

binds to

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39
Q

DNA regulatory elements refer to

A

DNA sequences known to be bound by transcription factors that activate or repress transcription

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40
Q

methods of introducing transgenes into an animal

A

direct injection
chemical transfection
electroporation
infection

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41
Q

chemical transfection transgene delivery involves

A

Incubate cells in culture medium containing DNA and a chemical that wraps the DNA up and is either endocytosed or that diffuses through the cell membrane

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42
Q

to increase the chance of the transgene being carried by all the cells we introduce it to the

A

one cell embryo (the fertilised zygote) otherwise you get a mosaic

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43
Q

direct injection delivery of a transgene is most effective when you target

A

male pronucleus after fertilisation of oocyte but before nuclear fusion

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44
Q

nuclear processing and free DNA

A

Repair mechanisms presumably recognise ‘free’ DNA in nucleus and try to do something with it

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45
Q

nuclear processing and our transgene

A

May randomly accidentally introduce breaks in host DNA and ligate transgene

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46
Q

When the transgenic animals are made ⟶ what if expression is not as expected, what happened?

A

Weak promoter / Insufficient regulatory elements

Copy number (too large gets silenced by epigenetics)

Position effects (site of integration - random)

Epigenetic modification

Genetic background (farm animals, hence Dolly)

Very big transgenes (>1 Mb), which put the promoter of the transgene in its normal chromosomal context, usually work best

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47
Q

viral GAG gene is for

A

encodes proteins of nucleoprotein core of virion

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48
Q

viral POL gene is for

A

encodes reverse transcriptase, integrase etc functions

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49
Q

viral ENV gene is for

A

encodes surface protein components of virion

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50
Q

after getting the transgene packed up as an infective particle it is then

A

introduced into ‘helper’ cells

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51
Q

why is helper cells needed for viral transgene delivery

A

Packaging cells contain the genes encoding gag, pol, env, but do not have the packaging signal so the viral genes cannot be included in viruses

Instead, the gag, pol, env proteins package and make viral particles around the transgene DNA

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52
Q

are viral transgene delivery used to infect the whole animal?

A

no, Often used to introduce a gene into bits of the mid-gestation embryo to create a mosaic

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53
Q

viruses commonly used for gene viral delivery

A

Adenoviruses
Adeno-Associated Virus AAV
Lentiviruses

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54
Q

issues with adenovirus gene delivery

A

DNA genome
Gets eliminated by immune system (immune activation)
DNA genome

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55
Q

Adeno-Associated Virus AAV benefits

A

No pathogenicity
Can infect non-dividing cells
Can integrate - usually at AAVS1 site in Human chromosome 19
Popular for gene therapy

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56
Q

Lentiviruses is a

A

Retroviruses

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57
Q

benefits of lentiviruses

A

Very efficient infection, dividing and non-dividing cells

Stable integration - long term expression of transgene

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58
Q

why is long term expression with viral transgene delivery a problem?

A

Viral sequences are subject to silencing mechanisms

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59
Q

any side effects to viral transgene delivery?

A

recombination leads to infective new viruses

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60
Q

solution to a knockout gene mouse dying before the gene can be explored

A

conditional knockout/tissue specific knockout

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61
Q

tissue specific knockout uses

A

Cre/loxP or flp/frt

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62
Q

cre is a

A

A site-specific Recombinase Enzyme from the P1 phage

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63
Q

cre recognises

A

Recognises a 34 bp DNA sequence loxP

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64
Q

cre’s role

A

recognises two loxp segments in DNA, brings them together and then removes the excess lox p segment

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65
Q

principles of using cre/loxP conditional knockout

A

A line of mice in which loxP sites have been inserted around the gene of interest. The gene is said to be ‘floxed’.

and a different line of mice that express Cre recombinase from a tissue-specific promoter (e.g. directs Cre expression to pancreas only.

breed the two together

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66
Q

how to created floxed mice?

A

conditional targeting by homologous recombination in ES cells, inject into blastocysts, create chimeras, and breed

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67
Q

what is different about the homologous recombination when using lox P and FRT

A

post-homologous recombination the neomycin resistance gene can be removed to avoid it interfering with gene expression

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68
Q

how do you target cre expression to a tissue of interest?

A

Create a ‘Designer’ Promoter

or knock-in cre gene to the correct tissues in ES cells

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69
Q

why is an internal ribosome entry site necessary for a typical cre construct

A

necessary to ensure expression of cre and GFP/LacZ which enable you to verify the transgene delivery was successful

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70
Q

a typical cre construct then consists of

A

promoter, cre, IRES (internal ribosome entry site), GFP or LacZ, intron and SV40

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71
Q

how can we temporally control the effect of the cre-lox transgene?

A

Put the Cre gene on promoters that are responsive to drugs you can give the mice

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72
Q

what drugs can cre promoters be vulnerable to

A

Tetracycline

Tamoxifen

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73
Q

Tetracycline “tet-off system” Induction of Cre expression involves

A

The Tet-activator protein ⟶ Binds to the tet operon (tetO) to activate transcription of Cre
But not in presence of Tetracycline

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74
Q

tet-off tetracycline cre system without tetracycline results in

A

Tetracycline then withdrawn ⟶ TetA binds tetO ⟶ Cre is transcribed ⟶ floxed gene knocked out

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75
Q

tamoxifen induction of cre expression involves

A

Uses a fusion protein combining activity of Cre and a mutant form of the ligand binding domain of the estrogen receptor (ERTM)

Cre-ERTM does not bind estrogen, but does bind 4-OH-tamoxifen

In absence of Tamoxifen ⟶ Hsp90 grabs Cre-ER and keeps it in cytoplasm ⟶ no recombination in nucleus

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76
Q

upon addition of tamoxifen Cre-ERTM is

A

released from Hsp90, allowing access to the nucleus and Cre-mediated recombination

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77
Q

any issues with the cre-loxp system?

A

Not 100% efficient
Not reversible
Some evidence that high levels of Cre may be toxic to cells in some cases

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78
Q

3 mechanisms of genome editing include

A

zin finger nucleases
TALEN
cas9/CRISPR

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79
Q

the zinc finger is

A

Protein motif that binds ~3 bp of DNA

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80
Q

multiple zinc finger motifs in a protein can

A

give long,&raquo_space; 3 bp binding specificity to a transcription factor

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81
Q

normal physiological function of zing fingers are to

A

Zn-finger transcription factor binds specific consensus DNA sequences around genes and activates transcription

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82
Q

experimental zinc finger involves

A

the transcriptional regulatory domain is replaced by a DNA nuclease called FOK1

2 ZFN bring 2 FOK1 together and generate a double stranded break in DNA

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83
Q

2 ZFN bring 2 FOK1 together and generate a double stranded break in DNA is important because

A

Ds break will be repaired in the cell, but repair is ERROR-PRONE

Random bases inserted, or get small deletions or insertions as DNA repair enzymes join the two cut ends together

Makes mutations at the cut point with very high efficiency

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84
Q

the process by which zinc finger (ZFN) generate mutations is called

A

non-homologous end joining

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85
Q

TALENs stand for

A

Transcription Activator-Like Effector Nucleases

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86
Q

talens in principle work similar to

A

ZFNs

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87
Q

TALE’s are

A

highly conserved 33-35 bp repeat domains encoded by Xanthomonas spp. proteobacteria

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88
Q

TALEs consist of

A

a nonspecific FokI nuclease domain fused to a customizable DNA-binding domain

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89
Q

TALEN method of Non-homologous End Joining Repair is

A

2 TALENs binding specific sequence of target gene to create double strand break

2 hypervariable amino acids determine which base each TALE repeat binds to

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90
Q

Cas9 is

A 
An endonuclease (cuts DNA) 
From bacterial acquired immunity against viruses
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91
Q

cas9 is based on

A

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

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92
Q

cas9 is targeted to cleave DNA by

A

short RNAS:
CRISPR-RNA (crRNA) and transactivating CRISPR-RNA (tracrRNA)

Are complementary to the site to be cut

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93
Q

Cas/CRISPR system can cut any piece of genomic DNA that is adjacent to a

A

‘PAM’ (Protospacer adjacent motif)

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94
Q

cas 9 PAM is

A

NGG

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95
Q

the first stage of CRISPR/mas mutagenesis then involves

A

The first CRISPR crRNA has a region that is complementary to part of the target gene, upstream of the PAM

It wriggles into the DNA duplex and binds by base-pairing

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96
Q

once the CRISPR crRNA has bound upstream to PAM then

A

The transactivating CRISPR tracrRNA has a region that is complementary to the rest of the crRNA
It binds by base-pairing

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97
Q

once cas9 binds to the CRISPR crRNA/ tracrRNA complex

A

cas9 will make a double-stranded break in the target DNA, a variable number of bases from the PAM
Break will be ragged, with single strand overhangs

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98
Q

the consequent break by cas9 stimulates

A

DNA repair enzymes will try to repair the damage

They will screw it up, creating a mutation

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99
Q

cas9 then generates mutations through

A

non-homologous end joining

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100
Q

in the lab crRNA-tracrRNA are fused into

A

sgRNA

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101
Q

Targeted ES cells with Cas9, and sgRNAs against multiple alleles of a gene was found to

A

With high efficiency, produced ES cell clones with mutations in all 6 alleles of all three genes

to quickly target ES cells with multiple mutations and make mutant mice

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102
Q

When Cas9 was injected into 1 cell zygotes what percentage of offspring had the mutations against TET genes?

A

50-90%

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103
Q

a limitation of using cas9 in 1 cell zygotes were

A

mutations induced are Uncontrolled and Variable

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104
Q

how may cas9 Uncontrolled and Variable mutations be overcome?

A

Catalyse some Homology-Directed Repair (HDR

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105
Q

Homology-Directed Repair (HDR) involves

A

Electroporate or inject cells with Cas9, sgRNA AND a single-stranded DNA oligonucleotide homologous to the area around the breakpoint

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106
Q

can Homology-Directed Repair be used alongside TELNS or zinc fingers?

A

yes

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107
Q

Cas9 + HDR can be used to introduce what other form of genetic manipulation into 1 cell zygotes?

A

loxP sequences

108
Q

any examples of Cas9 + HDR + loxP being used

A

Floxed the Mecp2 gene

that is mutated in Rett Syndrome

109
Q

limitations of cas9 are

A

Off-target mutations of Cas9/CRISPR remain a problem (especially for human therapy).

110
Q

how to clone an animal?

A
  1. Take a cell from the donor animal to be cloned
  2. Take the nucleus (pronucleus) out of a recipient unfertilised oocyte
  3. Put the nucleus of the donor somatic (e.g skin) cell into the enucleated oocyte
  4. ‘Activate’ the oocyte to start its development
  5. Transfer it to the uterus of a pseudopregnant female
  6. Completes development as normal in utero
111
Q

why does any cell work for cloning?

A

Every Cell Contains a Complete Genome

112
Q

how can an oocyte be activated to start its development?

A
  • electrical or chemical activation
113
Q

for cloning how can you put the nucleus of the donor somatic (e.g skin) cell into the enucleated oocyte

A

direct injection or electrofusion

114
Q

any examples of chemical activation for oocyte development?

A

strontium chloride will stimulate the calcium activation wave

115
Q

issues with cloned animals include

A

Dolly died young of lung disease
⟶ Viability of cloned embryos very low
⟶ ‘Large offspring syndrome’ common
⟶ Respiratory and circulatory problems common
⟶ Weak immune system
⟶ Liver failure
⟶ Premature ageing (including arthritis))

116
Q

why do issues arise with cloned animals?

A

⟶ Failure of epigenetic reprogramming (some retention of DNA character of donor cells) – this is a technical not a biological hurdle.
⟶ Accumulation of damage macromolecules – should be diluted out
⟶ Shortened telomeres - yes in sheep and goats, not in all species
⟶ Retention of mutations that happened during life of donor – very likely to happen.

117
Q

can serial cloning for many generations work?

A

yes for mice!

118
Q

what is the possible pathway for human cloning?

A

skin sample->inject into host oocyte and make blastocyst clone->harvest ICM and make ES cells->differentiate into necessary cells, tissues etc

119
Q

what is important about this process of human cloning (protocol for dopamine neurone production e.g.)

A

follows a stepwise process that mimics using external chemical influences actual human physiological differentiation and maturation

120
Q

can these cloned cells, such a dopamine neurones be grafted onto animals?

A

yes and survive

121
Q

did the grafting of human cloned dopamine neurones onto PD animals yield any improvements?

A

yes

122
Q

stem cell technology challenges

A

clinical grade culture facility free of pathogens

preventing spontaneous differentiation

animal product free mediums (risk of virus and immunity)

to minimise tumorigenic potential of transplants (teratoma)

overcome host immunity

produce all cell types necessary

123
Q

Adult somatic cells can be induced to become pluripotent by forced expression of a few key transcription factors such as

A

c-Myc, Oct4, Sox2, Klf4

124
Q

these induced pluripotent stem cells were verified by demonstrating

A

Teratoma formation with differentiated cells from all three embryonic layers

forming embryoid bodies (clumps of differentiated cells)

and could produce chimera

125
Q

Oct4, Sox2 (with Nanog) function as .

A

core transcription factors in maintaining pluripotency

126
Q

C-Myc is an

A

An oncogene, enhances proliferation

127
Q

C-Myc use in IPSC may be

A

Associates with histone acetyltransferase complexes responsible for opening up DNA for transcription. Possibly allows Oct4 and Sox2 to bind target loci.

128
Q

Klf4 use in IPSC may be

A

represses p53 which in turn represses Nanog (may therefore be back door to induction of endogenous Nanog).

129
Q

Tetraploid cells in pregnancy can

A

can contribute to the placenta but not to the embryo proper.

130
Q

injecting iPS cells into a tetraploid mouse blastocyst and transferring the blastocyst to the uterus of a recipient female to complete pregnancy, Boland et al were able to create

A

adult mice derived entirely from iPS cells

131
Q

other alternative transcription factors for producing ISPC

A

OCT4, NANOG, SOX2 and LIN28

132
Q

any proof of therapeutic use of ISPCs?

A

Treatment of Sickle Cell Anemia mouse model with iPS cells generated from autologous skin.

133
Q

Treatment of Sickle Cell Anemia mouse model with iPS cells generated from autologous skin limitations when considering humans?

A

Paper ends with a caveat that this is not suitable for humans because of the requirement to use retroviruses (risk of insertion mutagenesis) and oncogenes (c-Myc).

134
Q

when producing differentiated stem cells, in comparison to ES-cells, iPS cells were

A

dramatically less efficient at forming haemangioblasts, significantly more apoptotic, and with severely limited growth and expansion capacity

135
Q

why were IPS cells limited in their ability to differentiate

A

Induction of pluripotency requires somatic cells to lose their epigenetic memory

iPS cells retain some of the methylation pattern of the somatic cells from which they were derived, and are more prone to differentiate back into those same sorts of cells than into new cell types

136
Q

what percentage of AD is familial

A

1%

137
Q

BACe1 codes for

A

beta secretase

138
Q

an ideal gene delivery system should

A

transfer genes into both developing and mature animals;

transduce cells with high efficiency;

be cell-type / tissue specific;
mediate high level, long-term expression;

cause limited cytotoxicity;

elicit a small/negligible immune response;

allow sufficient lengths of DNA to be introduced;

avoid over-expression;

allow regulatable expression

139
Q

Novel viral models of Alzheimer’s disease enable studies on

A

Cellular mechanisms of degeneration
Drug target identification
Initial evaluation of treatments
On demand delivery to many species and preparations,
hence addressing the 3Rs of animal experimentation

140
Q

problems with transgenic mice models when modelling AD and using multiple genes

A 
Varied promoters
Varied genetic backgrounds
Technical uncertainties 
No suitable controls
Difficult to compare
141
Q

PLB mice for understanding AD were produced using

A

Knock-in (vs pronuclear injection)

142
Q

the benefits of the PLB mice model for AD were

A

Safe, removable, single-copy, stable genetics
Comparable triple / double / single lines [APP, Tau, PS1 & BACE]
Low / relevant expression levels (model of prodromal dementia)

Ideal to develop sensitive, early & translational biomarker

143
Q

BACE is rate limiting step in production of

A

b-amyloid

144
Q

rational for the production of PLB 4 hBACE1 knock in model

A

No hBACE tg mouse available
BACE is rate limiting step in production of b-amyloid
hBACE1 allows improved drug discovery
Model does not rely on rare genetic mutations

145
Q

PLB 4 hBACE1 knock in mice model pathological findings for AD were

A

reduced full length APP

accumulation of APP cleavage products

severe inflammation in the hippocampus and cortex

146
Q

PLB4 BACE1 knock-in mice: Behavioural characterization

A 
reduced:
Activity 
Spatial memory
Working memory  
Semantic memory

Anxiety

Age-dependent
AD-relevant

147
Q

did hBACE 1 mice models demonstrate any systemic effects despite being specific for neuronal tissue?

A

PLB4 mice are susceptible to diabetes, high fat diet

A glucose tolerance test, determine their fasting glucose level and then see how fast they can cleave glucose from their system. PLB4 much higher glucose level at 5 month

148
Q

BACE1 may be a link between what two pathological conditions

A

between AD and T2D

149
Q

what hypothalamic gene expression profiles were altered in PLB4 mice

A

changes related to appetite & energy regulation, a dramatic up-regulation of CHOP (multifunctional & pro-apoptotic transcription factor)

CHOP reflects ER stress

150
Q

how may BACE1 relate to ER stress

A

factors induce ER stress, this results in a signalling molecule being sent to activate stress response genes and alter translation necessary for plasticity. it is believed inflammation and BACE1 can interact with this pathway increasing it and thus altering plasticity and triggering apoptosis & autophagy

151
Q

factors causing ER stress

A

Ageing (mitochondria)
Obesity / Metabolic failure
Excitotoxicity
Ca2+ dyshomeostasis

152
Q

symptoms of parkinsons

A

Tremor
Ridigity
Akinesia
Postural instability

(TRAP)

153
Q

indirect symptoms of parkinsons

A

Depression
Dementia
Fatigue
Apathy

154
Q

ideal model of PD should include

A 
Pathological and clinical features
Dopaminergic and nondopaminergic systems
Central and peripheral nervous system
Motor and non-motor symptoms
Age-dependent onset and progressive nature
155
Q

are there any natural models for PD?

A

no

156
Q

in silico models for PD are useful for

A

Drug candidates:
Design
Screening

Mathematical models:
Metabolomics

157
Q

in vitro models of PD are useful for

A

Disease mechanism on cellular level:
Uptake of α-synuclein
Seeding capacity

Screening of drug candidates:
Reaction to molecules on cellular/organ level

158
Q

in vivo non mammalian models for PD are useful for

A

Genetically easily manipulated:
Understanding basis of pathogenesis
Molecular interactions
Mitochondrial studies

Less ethical issues

Drug discovery

159
Q

what is the limitation of in vivo non-mammalian models

A

99% of PD is non-hereditary

160
Q

benefit of mammalian models of PD

A

Validation of in vivo
Complex organism
Behaviour

161
Q

what percentage of DNA is shared between mice and men

A

97.5%

162
Q

gold standard mice neurotoxic model of PD

A

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model

163
Q

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model works by

A

Mitochondrial inhibition in nigrostriatal pathway:

Oxidative stress -> loss of dopaminergic neurons

164
Q

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model pathology

A

PD-like motor symptoms

Lacks formation of Lewy bodies

165
Q

can 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model pathology be injected across the BBB

A

easily and systemically

166
Q

key drugs developed using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model were

A

Levodopa
MAO inhibitors
COMT inhibitors

167
Q

an alternative neurotoxic model of PD is

A

6-hydroxydopamine (6-OHDA) model

168
Q

6-hydroxydopamine (6-OHDA) model is produced by

A

Injected into the substantia nigra or striatum

169
Q

6-hydroxydopamine (6-OHDA) models specifically

A

Modelling motor symptoms, not disease etiology

170
Q

6-hydroxydopamine (6-OHDA) model produces

A

Oxidative stress
Neurodegeneration
Neuroinflammation
Neuronal death by apoptosis

171
Q

PD mouse model transgenes are

A

LRRK2, SNCA

PINK1, PRKN, PARK7

172
Q

PD mouse model transgene features

A

Molecular pathways
No or little neurodegeneration
No Lewy body formation

173
Q

α-synuclein spreads through the brain according to the

A

Prion-hypothesis

174
Q

Mouse models of PD: Seeding of α-synuclein involve

A

Injection of human α-synuclein directly into brain which stimulate murine alpha synuclein to propagate and become pathological

175
Q

techniques to study mice behaviour include

A 
catwalk
rotarod 
elevated plus maze
agora
open field 
morris water maze
y maze
176
Q

behaviour affected by neurotoxic PD models?

A

yes, heavily

177
Q

behaviour affected by transgenic PD model

A

not at all

178
Q

behaviour affected by seeding PD model

A

yes mild

179
Q

cognition affected by neurotoxic PD model

A

yes

180
Q

cognition affected by transgenic PD model

A

not at all

181
Q

cognition affected by seeding PD model

A

yes

182
Q

social behaviour affected by transgenic PD model?

A

not at all

183
Q

social behaviour affected by seeding PD model?

A

yes, mild

184
Q

equation for energy balance

A

(ES)=(EI)-(EO)

185
Q

ES stands for

A

Rate of exchange in the body stores

186
Q

EI stands for

A

Chemical energy from the diet

187
Q

EO stands for

A

Work and heat

188
Q

(EO) or work and heat energy balance can be divided into

A 
Resting Metabolic Rate
(RMR)
Physical Activity
(PA)
Diet-induced Thermogenesis (DiT)
Thermogenesis
189
Q

what does diet really reflect?

A

bioavailability rather than just intake

190
Q

factors of bioavailability include

A 
Food addictions
Nutrient density &
quality
Allergies/Intolerances
Digestive Tract Diseases
Hunger/Satiety
191
Q

energy expenditure by resting metabolic rate has to consider

A

body composition (fat, fatre free mass, TBW, bone, organs etc)

192
Q

does base metabolic rate decrease with age?

A

yes, but plateaus around 20 rather than continuing to decrease

193
Q

what are the two central regulators of energy balance

A

hindbrain and hypothalamus

194
Q

what hypothalamic nuclei regulates energy balance

A

arcuate nucleus (ARC)

195
Q

arcuate nucleus (ARC) receives inputs from

A

grehlin, satiety peptides, leptin and insulin

196
Q

hindbrain nucleus receives inputs from

A

vagal afferents (stretch, chemo, nutrient)

197
Q

the hind brain nucleus is

A

nucleus tractus solitarius

198
Q

central regulation of energy balance outputs are

A

meal timing
meal volume
gastric emptying
energy expenditure

199
Q

benefits of mice models when considering obesity models

A
  1. Mouse obesity and metabolic phenotypes are comparable to humans
  2. Phenotype measured with standardized test
  3. Easy study of the brain
  4. Manipulate their genome
  5. Use identical strains when inbreeding
  6. Control of environmental factors
  7. Accelerated lifespan 1/30
  8. Cost-effective and efficient tool
200
Q

3R’s of animal research

A

replacement, reduction, refinement

201
Q

Replacement -

A

Methods which avoid or
replace the use of
animals

202
Q

Reduction -

A

Methods which minimise
the number of animals
used per experiment

203
Q

Refinement -

A

Methods which minimise
animal suffering and
improve welfare

204
Q

what is important to consider about the gross anatomical arrangement of the mouse brain and energy circuit

A

the energy circuit is close to the centres for decision making, motivation and arousal with several links

205
Q

environmental models for studying obesity use

A 
Maternal and early-life diet
Scheduled meal events
Fat/sugar choice diets
Binge-type feeding
Cafeteria/Junk diets
206
Q

chemical models for obesity use

A

Glucocorticoids
Streptozotocin
Antipsychotics

207
Q

chemical models for obesity - Glucocorticoids

A

Adipocyte differentiation,
lipolysis, proteolysis, gluconeogenesis. Induce
food intake, weight gain, adiposity, glucose
impairment.

208
Q

chemical models for obesity - Streptozotocin

A

Chemical ablation of pancreatic b-cells.

Model of diabetes

209
Q

chemical models for obesity - Antipsychotics

A

Fat deposition, FFAs

210
Q

pros of environmental/chemical models

A

Suitable for the investigations of non-genetic lifestyle dependent metabolic syndrome in humans

Inexpensive

211
Q

cons of of environmental/chemical models

A

Sometimes not complete reproducibility

Lack of standardized diets

Delayed onset of metabolic syndrome

212
Q

the leptin receptor when bound by leptin

A

inhibits appetite

213
Q

a single molecule, have been crucial to understand their role
in the pathophysiology of obesity. ie

A

leptin

214
Q

3 forms of genetic models for studying obesity

A

Spontaneous mutations
Artificially-induced mutations
Targeted mutations.

215
Q

Artificially-induced mutations limitations

A

Not targeted mutation

Need to screen for all genome and study whether it causes a phenotype

Expensive

216
Q

leptin acts through what signalling molecule

A

STAT3 signalling

to regulate adiposity

217
Q

Mutation in STAT3 (s/s) shows

A

the same overweight phenotype, but
glycemia and insulin sensitivity is
corrected under food restriction.

218
Q

conventional GMO mice model limitations

A

Knocking-out a gene can create early embryonic death
preventing the study in adults

Since the animal develops and grows without a specific gene,
compensatory mechanisms can appear.

219
Q

what alternative GM Advanced technique allows to interrogate genes
in specific tissues, cell type and even at precise times.

A

Cre/LoxP > Flp/Frt > Dre/Rox systems

220
Q

an example of mice obesity study using cre/LoxP

A

Mouse InsRLoxP

insulin receptor insR

221
Q

Recombinant virus targeting in cre mouse models for obesity study enables

A

long-term, cell specific and spatio-temporally genetic access to
certain cell populations

222
Q

what three areas in regards to neurones can viral particle and cre mouse models allow us to study

A

neuronal effect, connection effect and mapping -

with anterograde and retrograde tracing

223
Q

chemo genetics requires

A

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)

224
Q

how does DREADDs work

A

using viral particles and cre mouse models to insert receptors that are only activated by specific drugs to assess function of specific neurones in specific brain regions

225
Q

Opsins are

A

light-gated ion channels or pumps that absorb light at specific
wavelengths. Upon activation by light, these channels and pumps respond by
opening or closing, which conducts the flow of ions into or out of the cell

226
Q

Optogenetics enable the ability to

A

modulate terminal projections to evaluate target regions in real time

227
Q

Photometry uses

A

Calcium imaging enables neuroscientists to visualize the activity of hundreds
of individual neurons simultaneously using fluorescent activity sensors.
Changes in fluorescence indicate fluctuations in intracellular calcium, which is
an indirect indicator of neural activity

228
Q

Chemogenetics revealed AGRP neurones in arcuate nucleus (ARC)

A

activation of AgRP neurons induce food intake and

body weight gain

229
Q

Optogenetics revealed AGRP neurones in arcuate nucleus (ARC)

A

Activation of AgRP neurons do not induce food intake through the parabrachial nucleus

230
Q

photometry revealed AGRP neurones in arcuate nucleus (ARC)

A

AgRP neurons drive only the initiation of the meal event

231
Q

channelrhodopsin is both

A

fluorescent and an ion channel

232
Q

in response to blue light the channelrhodopsin channel

A

opens, Na+ and Ca2+ ions flow in, the nerve cell depolarizes and fires action potentials

233
Q

halorhodopsin is a

A

light-gated chloride pump found in archae

234
Q

halorhodopsin in responds to what colour of light

A

yellow

235
Q

halorhodopsin in response to yellow light

A

activates halorhodopsin pump, chloride enters, nerves hyperpolarise, silences action potentials

236
Q

the light sensitive protein channelrhodopsin is harvested from

A

algae

237
Q

together channelrhodopsin and halorhodopsin allow

A

multiple-color optical activation, silencing and desynchronization of neural activity.

238
Q

Optogenetic modulation occurs at three levels of neuronal activity:

A

1) light activated signal transduction cascade
2) light mediated intracellular protein-protein interactions
3) light switchable transgene for control of gene expression

239
Q

what are ospin receptor chimeras?

A

G-protein channel receptors combined with rhodopsin

240
Q

what is the intracellular loops of rhodopsin replaced with in chimeric receptors

A

specific adrenergic or serotonin receptors

241
Q

chimeric receptors enable manipulation of

A

alpha 1 adrenargic receptor - IP3, DAG

beta 2 adrenergic receptor - cAMP

242
Q

Optogentics + pharmacology can allow the study of

A

signalling - protein-protein interactions

243
Q

LightOn, uses the light dimerization property of Vivid to produce a

A

a light oxygen voltage domain containing protein in circadian clock systems, to form a synthetic light-switchable gene-promoter system.

244
Q

LightOn thus then can

A

binds promoters after blue light exposure

rapidly initiate transcription of target transgenes in both mammalian cells and mice

Giving precise spatiotemporal control of genes in a cell type–specific fashion - with light

245
Q

Therapeutic uses for optogenetics

A

gene therapy - install light-sensitive opsins in nociceptor nerve endings mouse skin

246
Q

in therapeutic optogenetics blue light causes

A

pain

247
Q

in therapeutic optogenetics yellow light causes

A

blocks pain

248
Q

Channelrhodopsin can be used to tranfect what to achieve potential therapeutic effect

A

retinal ganglion cells in humans blinded by retinitis pigmentosa - restore vision

249
Q

the connectome project aims to

A

mapping the entire connections of the human brain

250
Q

the connectome project uses

A

loxP and cre recombinase system

251
Q

why can cre/LoxP generate different colours for the brainbow

A

Cre/LoxP system can be used to introduce different colour tags with some being removed. Furthermore, whilst two forward facing LoxP sections will result in excision, two loxP sections facing one another will cause inversion of the code

252
Q

what are the four initial cre-lox P brainbow colours

A

red, green, yellow, blue

253
Q

how does a rainbow be generated from LoxP however

A

expression of trimeric combination of different fluorescent proteins and different numbers of copies result in different intensity

254
Q

how many colours are available for the brainbow project

A

ten

255
Q

has the brainbow project produced any revelations?

A

challenges concept of action potential dependent synapse withdrawal in developing visual system -

See clusters of retinal terminals from >10 uniquely labeled RGCs make complex retinothalamic synapses

Synapses are not withdrawn – lots of convergence persists on thalamic relay cells.

256
Q

the goal of the clarity project is to

A

creates see through brain

travel through brain structures in 3D, dynamically

label lots of molecules in whole brain

trace single cells through a forest of other cells

257
Q

is clarity just limited to the brain

A

no, other organs can undergo

258
Q

clarity process involves

A

infuse mouse brain with a hydrogel solution.
set hydrogel mesh binds and supports rest of brain
gel does not bind lipids or fats
they need removed as they are opaque
extract unbound fat - leaves clear view of everything else
proteins embedded in cell membranes and
dendritic spines on neurons - both remained.
Add brainbow labels

259
Q

has clarity been used to demonstrate anything?

A

Mice given cocaine, or electric shocks + Clarity + dyes to highlight cells that fired

shows which networks of neurons corresponded to pleasure, or to fear

both emotions are laid down in the medial prefrontal cortex

but stored along different pathways or axonal projections

And they are connected to different regions in the brain

260
Q

the results of the clarity pleasure and fear study

A

both emotions are laid down in the medial prefrontal cortex

but stored along different pathways or axonal projections

And they are connected to different regions in the brain

261
Q

Algebraic Topology seeks to

A

describe emergent behaviours of neural networks from their underlying structural firing patterns – single cells, groups, networks, synchrony/asynchrony

262
Q

according to Algebraic Topology neurones

A

tend to form families (called “cliques”), where each neuron is connected to each other in the clique

263
Q

according to algebraic topology the model brain reacts by

A

building and then collapsing a multi-dimensional connected tower

264
Q

what are some of the controversies regarding algebraic topology

A

some accuse it of lacking any substantial data, using too much jargon and having grandiose claims

265
Q

algebraic topology grandiose claims include

A

being able to code for decision making in brains and if correct, one day, be able to stimulate decisions in brains

neuroscience (2 decks)

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