From past to future: how did neuroscience begin and where is it taking us?

Question 1

What is the basic definition of neuroscience?

& the object of it?

Answer 1

the study of the brain and the nervous system in health and disease

with the object of understanding the functions of the nervous system at many levels of analysis (molecular, cellular, synaptic, network, computational and behavioural approaches).

Question 2

What is trepanation? & what is the modern version used for?

Also explain the cult following

Answer 2

Pre historic brain surgery

Trepanation is thought to have been an early conceptualisation for a disease or mental illness.

A modern version of this technique is used to release pressure when there is intracranial swelling.

There is a modern cult following of trepanation, whose purpose is to achieve higher levels of consciousness, although some argue it is just a new form of self-harm

Question 3

Explain the Edwin Smith surgical papyrus?

Answer 3

oldest known medical writing in history

featured many sophisticated observations about the brain and its connections

Question 4

What were the Greek contributions to medicine? & what was the key advice given?

Answer 4

Greek medicine moved towards observation and logical reasoning.

changes in diet, beneficial drugs and keeping the body in balance.

Question 5

What was the humoral theory of health, include the 4 fluids?

Answer 5

blood, phlegm, yellow bile and black bile

• needed to be kept in balance for wellness. Illness was supposedly caused when these fluids became out of balance, and sometimes required an intervention in order to set things righ

Question 6

Rene descartes

What is the concept of dualism?

Answer 6

the brain and the body are mechanical things but the mind is not physical and, therefore, not subject to scientific observation.

This influenced our thinking on animals, leading to several experiments being carried out, which led to much of the understanding in neuroscience we have today.

Question 7

What is the concept of monism?

Answer 7

the mind is a product of physical neural activity.

Question 8

In 1664, Thomas Willis publishes The Anatomy of the Brain, the first book really dedicated to the study of the brain.

What does it describe? & what term is used for the first time?

Answer 8

It described reflexes, epilepsy, apoplexy and paralysis, and for the very first time, the term ‘neurology’ is used.

Question 9

Who was the father of microscopy?

Answer 9

Anton van Leeuwenhoek
(1632-1723)

Created the very first microscope in 1674.

Question 10

Who established electricity as the mode of communication used by the nervous system?

Answer 10

Luigi Galvani

Question 11

Mary Shelly, using the idea that electricity could reanimate dead tissue, she wrote what?

Answer 11

Frankenstein

Question 12

What is the Bell Magendie Law?

Answer 12

representation of division of labour within the brain.

first time it was figured out that information travels in 1 direction, not 2 along motor and sensory nerves

Question 13

Charles Darwin and Russell Wallace created new concepts of what?

Answer 13

• evolution
• common traits between humans and
animals (such as the brain)

Question 14

What is the ‘Science of phrenology’?

Answer 14

non-invasive attempt that observes the structure (observable bumps) of a person’s skull, and then correlate them with all sorts of personality characteristics and abilities

• incorrect however very popular at the time (in the 1970s)

Question 15

What did phrenology, although incorrect pave the way for?

Answer 15

Localisation of function

Question 16

What neuroimaging technique was thought to be compared to phrenology?

Answer 16

fMRI

Question 17

What was Paul Broca’s influence?

Answer 17

Used clinical observation with autopsy to study the correlation between brain damage and behaviour deficits.

Question 18

What is the Broca’s area linked to?

Answer 18

linked to speech production and language functions

Question 19

What did Gustav Fritsch and Eduard Hitzig accomplish regarding in vivo experimentation on animals?

Answer 19

Used in vivo experimentation on animals to demonstrate the localisation of motor function and the property of contralateral control when stimulating the motor cortex.

Question 20

Explain the neuron doctrine with regards to Camillo Golgi and Santiago Ramón y Cajal? and why they disagreed with each other?

Answer 20

Golgi believed that the nervous system was neural net, a vast network of continuous fibres.

Cajal argued that the nervous system was an array of discrete independent cells, now known as the neuron doctrine:
a brain made of independent cells that talk to one another

• Golgi created Golgi stain
• Cajal, in turn, used the Golgi stain to prove his theory of individual cells.

Question 21

Explain Otto Loewi’s Vagusstoff experiment in 1921 (chemical transmission)?

and what was the chemical used to prove this?

Answer 21

This was the first piece of evidence to show the
importance of chemicals in neural transmission which, along with the critical element of electricity, leading us to say that neurons are electrochemical in nature.

chemical used is acetylcholine

Question 22

Who coined the word synapse?

Answer 22

Charles Sherrington

Question 23

TOPIC 2: NEURAL STEM CELLS

Question 24

What is the definition of neural stem cells?

Answer 24

Undifferentiated cells, defined by their capacity for self-renewal and multipotency

Question 25

During central nervous system development, neural stem cells proliferate and divide to generate clonally related progeny that differentiate into what?

Answer 25

neurons, astrocytes and oligodendrocytes

Question 26

Symmetric division of neural stem cells include?

Answer 26

• ability to self-renew • maintains neural stem cell (NSC) population

Question 27

Asymmetric division of neural stem cells include? And what lineages are they restricted to?

Answer 27

* produces one NSC, and one neural progenitor cell (NPC) | * restricted to neuronal or glial lineages

Question 28

what are the 3 types of stem cell potencies?

Answer 28

1.Totipotent ‘Toti’ = whole Can give rise to any and all human cell types and to an entire functional organism Most versatile stem cell 2. Pluripotent "Pluri"= many Can give rise to any and all human cell types Day four of development: embryo forms into two layers, outer layer and inner mass. As pluripotent stem cells continue to divide, they begin to specialise further 3. Mulitpotent "multi"= several Can give rise to a limited range of cells within a tissue type The offspring of the pluripotent cells become the progenitors of such cell lines as blood cells, skin cells and nerve cells

Question 29

What are examples of each?

Answer 29

totipotent: fertilised eggs (days 0-4) Pluripotent: inner cell mass layer of the blastocyst stem cells Multipotent: neural stem cells

Question 30

Neural stem cells are the source of all neuron types in the central and peripheral nervous systems True or False?

Answer 30

TRUE

Question 31

Neural stem cells from the neural tube are commonly named neuroepithelial cells, which means what? & in the cortex, neuroepithelial cells transition into what?

Answer 31

the most primitive form of neural stem cells. - radial glial cells, also a form of neural stem cells.

Question 32

How does the nervous system and neural stem cells begin forming? in 3 key areas

Answer 32

Primitive: CNS begins as a tube of neuroepithelia cells In the cortex: Neuroepithelial cells transition into radial glial cells. - this gives rise to neural progenitors, neurons, astrocytes and oligodendrocytes. In the spinal cord and striatum: Radial glial cells are not as prominent. Progenitors emerge from nonradial multipotent NSC populations.

Question 33

Once development has ended Neural stem cell populations are very rare. True or false?

Answer 33

TRUE

Question 34

What areas do the become restricted to once development has ended?

Answer 34

Neural stem cell populations are restricted to the dentate gyrus (DG) and the subventricular zone (SVZ).

Question 35

IDENTIFICATION OF NEURAL STEM CELLS

Question 36

To identify embryonic neural stem cells, it remains very controversial. There is a lack of very specific neural stem cell markers, which make it very difficult to demonstrate long-term self-renewal and multipotency in vivo. true or false?

Answer 36

TRUE

Question 37

How can the neural stem cells identity change?

Answer 37

The identity of NSCs could change spatially (neural tube position) and temporally (developmental age).

Question 38

What genes are among the earliest genes expressed in the early neural plate?

Answer 38

Sox genes (eg. Sox2)

Question 39

What 3 genes/proteins can be used as neural stem cell markers?

Answer 39

Sox2, nestin (intermediate filament protein) and mushashi (RNA-binding protein) can only be expressed in restricted progenitors

Question 40

No exclusive antigens have been identified for NSCs true or false?

Answer 40

TRUE

Question 41

Combinations of positive and negative markers are required to better identify NSCs TRUE OR FALSE?

Answer 41

TRUE

Question 42

One way to identify dividing cells, is to inject what?

Answer 42

bromodeoxyuridine or BrdU, which is an analogue of thymidine, that will intercalate in the DNA as it replicates Quantitative: we can count the number of cells that have been dividing

Question 43

Explain the Retroviral labelling technique for dividing cells? Is this quantitative or qualitative?

Answer 43

* retrovirus engineered to express GFP * injected locally in the hippocampus Qualitative: injected locally so we are not labelling the entire structure.

Question 44

SOURCES

Question 45

what are the sources of NSCs?

Answer 45

We can derive NSC from any time during development.

Question 46

Adipose stem cells have also been shown to not be potential candidates to generate neural stem cells. TRUE OR FALSE?

Answer 46

FALSE. they are potential candidates

Question 47

Explain the Multipotent lineage?

Answer 47

Generated from non-neural multipotent stem cells some evidence showing that bone marrow-derived mesenchymal stem cells and hematopoietic stem cells can give rise to neural stem cells

Question 48

Explain the differentiated lineage?

Answer 48

• reprogramming • using induced pluripotent stem cells (iPSCs) that then differentiate as NSCs

Question 49

What do we need to improve on? 6 areas If we want to fully exploit the potential of neural stem cells, we need to better understand how cells specialise.

Answer 49

1. Our understanding of cell specialisation 2. Our understanding of growth factors and gene regulation 3. Our understanding cell cycle control 4. We need to prove long-term stability of cells 5. We need to evaluate cell-host interactions 6. We need to build scientific capacity

Question 50

A tool for research

Question 51

We can collect stem/progenitor cells from three stages of development. What are the 3 stages?

Answer 51

1. late blastocysts 2. pre- differentiation (brain, olfactory system, spinal cord, blood and bone marrow) 3. damaged spinal cord

Question 52

Do endogenous stem/progenitor cells have direct or indirect transplantation?

Answer 52

DIRECT

Question 53

when are stem cells/progenitor cells transplanted?

Answer 53

after cell culture for propagation, pre-differientation or engineering

Question 54

Let’s look at the advantages and disadvantages of using various cell types for transplantation. Our situation: cell therapy in order to treat Parkinson’s disease. Explain the problem & the solution?

Answer 54

The problem: Degeneration of dopaminergic (DA) neurons in the substantia nigra (SN) The solution: Graft NSCs to produce DA neurons

Question 55

Explain further the process of Cell therapy for Parkinson’s disease: Foetal donor?

Answer 55

need to expand the cells in cell culture and immortalise them and then they could be transplanted to the patient

Question 56

What are the advantages of this process?

Answer 56

one time isolation from donor tissue required • foetal stem cells might have greater stability and plasticity

Question 57

What are the disadvantages of this process?

Answer 57

* ethics * immune suppression * mean of reversible immortalisation required * risk to patient

Question 58

* foetal brain is currently being used in a clinical trial for strokes * it has passed safety tests True or False?

Answer 58

TRUE, in phase 2

Question 59

Cell therapy for Parkinson’s disease: Blastocyst donor Explain this process?

Answer 59

We take the inner cell mass cells from the blastocyst, - derive embryonic cells from the blastocyst. - from the embryonic cell line, we need to derive neural stem cells - need to expand them before transplanting them to the patient.

Question 60

What are the advantages of this?

Answer 60

* can be used for therapy of other tissues (eg liver/heart) | * access to foetal tissue is not required

Question 61

What are the disadvantages of this?

Answer 61

* ethics * immune suppression * technical difficulties * time consuming

Question 62

Cell therapy for Parkinson’s disease: Somatic adult cells Explain this process?

Answer 62

Somatic adult cells (patient’s body cell such as hair) Derive iPSC line Derive NSC line Expansion Transplantation to patient

Question 63

Cell therapy for Parkinson’s disease: Adult brain Explain the process?

Answer 63

Adult brain biopsy Expansion in cell culture to immortalise. Transplantation to patient

Question 64

What are the advantages of this?

Answer 64

• access to foetal tissue is not required

Question 65

What are the disadvantages of this?

Answer 65

* access to donor tissue * risk to the donor * immune suppression * mean of reversible immortalisation required * risk to patient

Question 66

Cell therapy for Parkinson’s disease: in vivo mobilisation of endogenous brain stem cells with growth factors Explain this process?

Answer 66

The brain harbours neural stem cells and we potentially have the ability to mobilise these cells to repair the brain. - Inject growth factors intracerebrally - Mobilisation of endogenous stem cells

Question 67

what are the advantages of this?

Answer 67

* access to fetal tissue is not required | * no immune rejection

Question 68

what are the disadvantages of this?

Answer 68

• restricted adult NSC niches • could be a limited response • will these cells migrate and differentiate?

Question 69

What is the current use of in vivo mobilisation of endogenous brain stem cells with growth factors?

Answer 69

* currently being used in treating Parkinson’s * a clinical trial is ongoing * we await the results

Question 70

Topic 3: iPS cells and CRISPR

Question 71

what does CRISPR stand for?

Answer 71

clustered regularly interspaced short palindromic repeats

Question 72

what does CRISPR stand for?

Answer 72

clustered regularly interspaced short palindromic repeats

Question 73

CRISPR refers to the DNA sequences discovered in the genomes of what?

Answer 73

Bacteria These unusual DNA sequences were found to be part of a type of innate immunity that bacteria have developed against viruses.

Question 74

The CRISPR sequences in the bacterial genome arise because? 4 steps

Answer 74

1) bacterial cell is infected with viral infection 2) Viral DNA is processed by the bacteria into short segments 3) short segments are integrated into the host genome 4) Bacteria then uses sequences of viral DNA to guide enzymes, called a nuclease to destroy any viral infection

Question 75

This is highly specific because?

Answer 75

1) the guide sequence is complimentary to the viral DNA 2) Upon binding the complimentary sequence to the viral DNA, the attached nuclease introduces a double-strand break in the DNA

Question 76

There are two other features of the CRISPR system that are important to mention regarding the nuclease CAS9 and the PAM sequence What are they?

Answer 76

(1) Nuclease used: typically CAS9 - cuts DNA with the guide sequence 4 nucleotides from PAM sequence (2) The PAM sequence for Cas9 is NGG, where N stands for any nucleotide - follows on directly after the guide sequence. - The PAM sequence is essential for binding of the CRISPR Cas9, but does not form parts of the guide sequence itself.

Question 77

Define Nonsense mediated decay?

Answer 77

the process of degrading abnormal RNA transcripts.

Question 78

What are two examples of the types of genetic changes that can be introduced using CRISPR to facilitate the study of disease in human cells?

Answer 78

(1) inactivating the function of a gene | (2) altering a single base pair to make a point mutation

Question 79

These two types of genome edits can be made by taking advantage of what?

Answer 79

two different repair pathways in the cell for fixing double-strand breaks in DNA. When a DNA strand undergoes a double-strand break, the cell typically uses one of two main pathways to repair it.

Question 80

Explain the 1st repair pathway?

Answer 80

1st: non-homologous end joining - the quickest and the most commonly used repair pathway in most cells, including pluripotent stem cells. However, this repair pathway is error-prone.

Question 81

How is the 1st repair pathway error prone?

Answer 81

- When a double-strand break is repaired by a non-homologous end joining, small sequences of DNA are sometimes added or removed to facilitate the repair process. - This change in the DNA sequence of the gene, and therefore the RNA transcript it produces, can be recognised by the cell as abnormal, and the transcripts are degraded before they can be translated into protein

Question 82

This process of degrading abnormal RNA transcripts is called nonsense-mediated decay, and if both alleles of a gene are mutated in this way what will happen to the gene?

Answer 82

the gene will be knocked out, in other words, unable to make a functional protein.

Question 83

How can CRISPR help in this situation?

Answer 83

CRISPR can therefore be used to relatively easily knock out genes of interest in human pluripotent cells. This allows us to understand the consequences of the loss of function of that gene.

Question 84

The second major repair pathway that a cell can use to repair a double-strand break is called?

Answer 84

homology-directed repair, so called because it uses a sister chromatid as a template to create a very accurate repair to the break

Question 85

When is this process active?

Answer 85

only active in a cell just before cell division, because this is the only time a cell has a second copy of its DNA close by to use as a template

Question 86

what is a Homology-directed repair (HDR)?

Answer 86

Insertion of a single stranded DNA molecule (100 base pairs)

Question 87

what are 3 Human diseases as a result of point mutation?

Answer 87

Parkinson's disease, Alzheimers and motor neuron disease

Question 88

How would you introduce a point mutation at a particular site in the genome?

Answer 88

- introduce the CRISPR with a guide to target and cut that particular site, along with a single-stranded DNA molecule complimentary to that region but carrying the point mutation. - The CRISPR will then cut the genomic DNA, and the cell will use the single-stranded DNA molecule containing the point mutation as a repair template. - The point mutation will therefore be incorporated into the genome at that precise location

Question 89

CRISPR technology in the treatment of amyotrophic lateral sclerosis (ALS)

Question 90

Define ALS?

Answer 90

ALS is a neurodegenerative disease characterised by the loss of motor neurons and progressive muscle wasting, with death typically occurring within three to five years of symptom onset.

Question 91

There are mutations found to be associated with ALS. What are some of the genes affected?

Answer 91

genes such as superoxide dismutase 1 (SOD1) TAR DNA-binding protein 43 - both single point mutations.

Question 92

What do some of the mutations cause?

Answer 92

a loss in function, whereas others are thought to give rise to a toxic gain-of-function, such as in the formation of toxic protein aggregates

Question 93

In cases of ALS, where point mutations are causing a toxic gain-of-function in the gene product, such as the formation of toxic aggregates, it may be sufficient to use what approach?

Answer 93

knock out a gene by creating a double-stranded break with CRISPR, leading to nonsense mutation and a rapidly degraded gene product. - to bring about an improvement in function - This assumes that the levels of expression of the gene can be reduced without serious adverse consequences.

Question 94

What are one of the main challenges in doing any gene therapy in the nervous system?

Answer 94

Getting CRISPR past the blood brain barrier

Question 95

What are the main advantages of the AAV vector for this? | And what specific serotype may be used?

Answer 95

it does not trigger a strong immune reaction in humans, and this particular viral serotype, serotype 9, can cross the blood-brain barrier and efficiently infects neurons after systemic administration

Question 96

Precise genome editing by homologous recombination: when is this approach particularly relevant?

Answer 96

where the gene is extremely dose-sensitive or where the mutation gives rise to a loss of function.

Question 97

One limitation of using AAV as a vector is what?

Answer 97

it cannot carry more than 4.5 kilobases of DNA

Question 98

What is the solution to this size issue?

Answer 98

package these components into two separate AAV vectors

Question 99

Homology-directed repair was shown in what percentage of the neurons in a range of brain regions?

Answer 99

10 %

Question 100

What is a major shortcoming of using CRIPSR in-vivo to knock out genes by introducing mutations or edit genes precisely using homology-directed repair?

Answer 100

the potential for off-target effects, such that other sites with a similar sequence within the genome are cut and mutated unintentionally.

Question 101

How can these off-target effects be minimised?

Answer 101

through bioinformatic design and testing. However, for clinical use, the possibility of such effects are highly undesirable

Question 102

What are 3 neurological diseases common to microsatellite repeat expansions?

Answer 102

* Huntington’s disease * fragile X syndrome * Certain forms of motor neuron diseases

Question 103

What are microsatellite repeat expansions

Answer 103

These are repetitive two to nine base pair sequences of DNA, often occurring in non-coding regions of protein coding genes. These sequences do occur in healthy individuals but are found to be greatly expanded in length in affected individuals

Question 104

How many Microsatellites repeat expansions do healthy humans have versus unhealthy?

Answer 104

Most healthy individuals (2-23 repeats of G4C2) Affected individuals (hundreds to thousands repeats) Increased risk of neurological diseases

Question 105

What do repeat expansions do?

Answer 105

* affect the normal expression of the gene * produce a toxic gain of function * make it impossible to eliminate their toxic effects by knocking out the host gene

Question 106

What is the possible treatment for this?

Answer 106

None so far, - produce a toxic gain of function - make it impossible to eliminate their toxic effects by knocking out the host gene - their size makes homology-directed repair impractical - possible treatment with two double strand breaks is associated with potential off-target effects and chromosomal abnormalities

Question 107

Adapting CRISPR technology to target RNA

Question 108

Targeting disease causing microsatellite sequences by using RNA and avoiding the risks associated with cutting genomic DNA. What is the method for this?

Answer 108

Adapting CRISPR to target those same repeat sequences at the RNA level

Question 109

What are the findings of this process?

Answer 109

CRISPR binds to and cuts this abnormal RNA leading to its degradation and the clearance of abnormal and toxic RNA foci in the cells.