From past to future: how did neuroscience begin and where is it taking us? Flashcards
What is the basic definition of neuroscience?
& the object of it?
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).
What is trepanation? & what is the modern version used for?
Also explain the cult following
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
Explain the Edwin Smith surgical papyrus?
oldest known medical writing in history
featured many sophisticated observations about the brain and its connections
What were the Greek contributions to medicine? & what was the key advice given?
Greek medicine moved towards observation and logical reasoning.
changes in diet, beneficial drugs and keeping the body in balance.
What was the humoral theory of health, include the 4 fluids?
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
Rene descartes
What is the concept of dualism?
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.
What is the concept of monism?
the mind is a product of physical neural activity.
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?
It described reflexes, epilepsy, apoplexy and paralysis, and for the very first time, the term ‘neurology’ is used.
Who was the father of microscopy?
Anton van Leeuwenhoek
(1632-1723)
Created the very first microscope in 1674.
Who established electricity as the mode of communication used by the nervous system?
Luigi Galvani
Mary Shelly, using the idea that electricity could reanimate dead tissue, she wrote what?
Frankenstein
What is the Bell Magendie Law?
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
Charles Darwin and Russell Wallace created new concepts of what?
• evolution
• common traits between humans and
animals (such as the brain)
What is the ‘Science of phrenology’?
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)
What did phrenology, although incorrect pave the way for?
Localisation of function
What neuroimaging technique was thought to be compared to phrenology?
fMRI
What was Paul Broca’s influence?
Used clinical observation with autopsy to study the correlation between brain damage and behaviour deficits.
What is the Broca’s area linked to?
linked to speech production and language functions
What did Gustav Fritsch and Eduard Hitzig accomplish regarding in vivo experimentation on animals?
Used in vivo experimentation on animals to demonstrate the localisation of motor function and the property of contralateral control when stimulating the motor cortex.
Explain the neuron doctrine with regards to Camillo Golgi and Santiago Ramón y Cajal? and why they disagreed with each other?
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.
Explain Otto Loewi’s Vagusstoff experiment in 1921 (chemical transmission)?
and what was the chemical used to prove this?
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
Who coined the word synapse?
Charles Sherrington
TOPIC 2: NEURAL STEM CELLS
What is the definition of neural stem cells?
Undifferentiated cells, defined by their capacity for self-renewal and multipotency
During central nervous system development, neural stem cells proliferate and divide to generate clonally related progeny that differentiate into what?
neurons, astrocytes and oligodendrocytes
Symmetric division of neural stem cells include?
• ability to self-renew
• maintains neural stem
cell (NSC) population
Asymmetric division of neural stem cells include?
And what lineages are they restricted to?
- produces one NSC, and one neural progenitor cell (NPC)
* restricted to neuronal or glial lineages
what are the 3 types of stem cell potencies?
1.Totipotent
‘Toti’ = whole
Can give rise to any and all human cell types and to an entire functional organism Most versatile stem cell
- 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
- 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
What are examples of each?
totipotent: fertilised eggs (days 0-4)
Pluripotent: inner cell mass layer of the blastocyst stem cells
Multipotent: neural stem cells
Neural stem cells are the source of all neuron types in the central and peripheral nervous systems
True or False?
TRUE
Neural stem cells from the neural tube are commonly named neuroepithelial cells, which means what?
& in the cortex, neuroepithelial cells transition into what?
the most primitive form of neural stem cells.
- radial glial cells, also a form of neural stem cells.
How does the nervous system and neural stem cells begin forming? in 3 key areas
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.
Once development has ended Neural stem cell populations are very rare.
True or false?
TRUE
What areas do the become restricted to once development has ended?
Neural stem cell populations are restricted to the dentate gyrus (DG) and the subventricular zone (SVZ).
IDENTIFICATION OF NEURAL STEM CELLS
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?
TRUE
How can the neural stem cells identity change?
The identity of NSCs could change spatially (neural tube position) and temporally (developmental age).
What genes are among the earliest genes expressed in the early neural plate?
Sox genes (eg. Sox2)
What 3 genes/proteins can be used as neural stem cell markers?
Sox2, nestin (intermediate filament protein) and mushashi (RNA-binding protein)
can only be expressed in restricted progenitors
No exclusive antigens have been identified for NSCs
true or false?
TRUE
Combinations of positive and negative markers are required to better identify NSCs
TRUE OR FALSE?
TRUE
One way to identify dividing cells, is to inject what?
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
Explain the Retroviral labelling technique for dividing cells?
Is this quantitative or qualitative?
- retrovirus engineered to express GFP
- injected locally in the hippocampus
Qualitative: injected locally so we are not labelling the entire structure.
SOURCES
what are the sources of NSCs?
We can derive NSC from any time during development.
Adipose stem cells have also been shown to not be potential candidates to generate neural stem cells.
TRUE OR FALSE?
FALSE. they are potential candidates
Explain the Multipotent lineage?
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
Explain the differentiated lineage?
• reprogramming
• using induced pluripotent stem cells (iPSCs) that then
differentiate as NSCs
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.
- Our understanding of cell specialisation
- Our understanding of growth factors and gene regulation
- Our understanding cell cycle control
- We need to prove long-term stability of cells
- We need to evaluate cell-host interactions
- We need to build scientific capacity
A tool for research
We can collect stem/progenitor cells from three stages of development. What are the 3 stages?
- late blastocysts
- pre- differentiation (brain, olfactory system, spinal cord, blood and bone marrow)
- damaged spinal cord
Do endogenous stem/progenitor cells have direct or indirect transplantation?
DIRECT
when are stem cells/progenitor cells transplanted?
after cell culture for propagation, pre-differientation or engineering
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?
The problem:
Degeneration of dopaminergic (DA) neurons in the substantia nigra (SN)
The solution:
Graft NSCs to produce DA neurons
Explain further the process of Cell therapy for Parkinson’s disease: Foetal donor?
need to expand the cells in cell culture and immortalise them
and then they could be transplanted to the patient
What are the advantages of this process?
one time isolation from donor tissue required
• foetal stem cells might have greater stability and plasticity
What are the disadvantages of this process?
- ethics
- immune suppression
- mean of reversible immortalisation required
- risk to patient
- foetal brain is currently being used in a clinical trial for strokes
- it has passed safety tests
True or False?
TRUE, in phase 2
Cell therapy for Parkinson’s disease: Blastocyst donor
Explain this process?
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.
What are the advantages of this?
- can be used for therapy of other tissues (eg liver/heart)
* access to foetal tissue is not required
What are the disadvantages of this?
- ethics
- immune suppression
- technical difficulties
- time consuming
Cell therapy for Parkinson’s disease: Somatic adult cells
Explain this process?
Somatic adult cells (patient’s body cell such as hair)
Derive iPSC line
Derive NSC line
Expansion
Transplantation to patient
Cell therapy for Parkinson’s disease: Adult brain
Explain the process?
Adult brain biopsy
Expansion in cell culture to immortalise.
Transplantation to patient
What are the advantages of this?
• access to foetal tissue is not required
What are the disadvantages of this?
- access to donor tissue
- risk to the donor
- immune suppression
- mean of reversible immortalisation required
- risk to patient
Cell therapy for Parkinson’s disease: in vivo mobilisation of endogenous brain stem cells with growth factors
Explain this process?
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
what are the advantages of this?
- access to fetal tissue is not required
* no immune rejection
what are the disadvantages of this?
• restricted adult NSC niches
• could be a limited response
• will these cells migrate and
differentiate?
What is the current use of in vivo mobilisation of endogenous brain stem cells with growth factors?
- currently being used in treating Parkinson’s
- a clinical trial is ongoing
- we await the results
Topic 3: iPS cells and CRISPR
what does CRISPR stand for?
clustered regularly interspaced short palindromic repeats
what does CRISPR stand for?
clustered regularly interspaced short palindromic repeats
CRISPR refers to the DNA sequences discovered in the genomes of what?
Bacteria
These unusual DNA sequences were found to be part of a type of innate immunity that bacteria have developed against viruses.
The CRISPR sequences in the bacterial genome arise because? 4 steps
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
This is highly specific because?
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
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?
(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.
Define Nonsense mediated decay?
the process of degrading abnormal RNA transcripts.
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?
(1) inactivating the function of a gene
(2) altering a single base pair to make a point mutation
These two types of genome edits can be made by taking advantage of what?
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.
Explain the 1st repair pathway?
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.
How is the 1st repair pathway error prone?
- 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
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?
the gene will be knocked out, in other words, unable to make a functional protein.
How can CRISPR help in this situation?
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.
The second major repair pathway that a cell can use to repair a double-strand break is called?
homology-directed repair, so called because it uses a sister chromatid as a template to create a very accurate repair to the break
When is this process active?
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
what is a Homology-directed repair (HDR)?
Insertion of a single stranded DNA molecule (100 base pairs)
what are 3 Human diseases as a result of point mutation?
Parkinson’s disease, Alzheimers and motor neuron disease
How would you introduce a point mutation at a particular site in the genome?
- 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
CRISPR technology in the treatment of amyotrophic lateral sclerosis (ALS)
Define ALS?
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.
There are mutations found to be associated with ALS. What are some of the genes affected?
genes such as superoxide dismutase 1 (SOD1)
TAR DNA-binding protein 43
- both single point mutations.
What do some of the mutations cause?
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
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?
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.
What are one of the main challenges in doing any gene therapy in the nervous system?
Getting CRISPR past the blood brain barrier
What are the main advantages of the AAV vector for this?
And what specific serotype may be used?
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
Precise genome editing by homologous recombination: when is this approach particularly relevant?
where the gene is extremely dose-sensitive or where the mutation gives rise to a loss of function.
One limitation of using AAV as a vector is what?
it cannot carry more than 4.5 kilobases of DNA
What is the solution to this size issue?
package these components into two separate AAV vectors
Homology-directed repair was shown in what percentage of the neurons in a range of brain regions?
10 %
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?
the potential for off-target effects, such that other sites with a similar sequence within the genome are cut and mutated unintentionally.
How can these off-target effects be minimised?
through bioinformatic design and testing. However, for clinical use, the possibility of such effects are highly undesirable
What are 3 neurological diseases common to microsatellite repeat expansions?
- Huntington’s disease
- fragile X syndrome
- Certain forms of motor neuron diseases
What are microsatellite repeat expansions
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
How many Microsatellites repeat expansions do healthy humans have versus unhealthy?
Most healthy individuals (2-23 repeats of G4C2)
Affected individuals (hundreds to thousands repeats) Increased risk of neurological diseases
What do repeat expansions do?
- 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
What is the possible treatment for this?
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
Adapting CRISPR technology to target RNA
Targeting disease causing microsatellite sequences by using RNA and avoiding the risks associated with cutting genomic DNA.
What is the method for this?
Adapting CRISPR to target those same repeat sequences at the RNA level
What are the findings of this process?
CRISPR binds to and cuts this abnormal RNA leading to its degradation and the clearance of abnormal and toxic RNA foci in the cells.
