Molecular neuropathology
INHOUD
• Introduction to Molecular Pathology
• Neuropathology – Brain Structure and Function
• Tools/techniques of Molecular Pathology (limited)
• Molecular Neuropathology of:
o Alzheimer’s disease, Parkinson’s disease, Lewy body disease, Frontotemporal dementia-ALS spectrum,
Stroke, Vascular dementia, Prion diseases
o Neoplasia (limited to glioblastoma)
o Select developmental disorders/ inborn errors
PowerPoint presentations on individual assignments (10 minutes each participant) – plenty of discussion
Make an interactive presentation - 3/4/5 slides min 6min max 10 account for 6 point of the 20
If he says it’s important for the exam mark the slide!!
Have to be very active in this course
PART 1 Introduction to Molecular Pathology
What is Molecular neuropathology?
Neuropathology = Pathology of the nervous system
Molecular neuropathology = Study at a molecular (biological)
level
we mostly deal with the protein part because we are interested
in the phenotype
this tells you how the field have not been collaborating too quickly because people are working on different things
2 fields have continued, the chromosome field has stopped quickly
what is the big deal about the DNA structure? you engaged public funding and research took a big jump from there on
So, what do we learn from Watson & Crick discovery?
• Dedication, dynamism, and creative thinking are critical in any discovery!
• But never undervalue basic concepts
• First Watson/Crick model had the phosphate backbones at the center (Linus Pauling also made the same
mistake) → P backbone cannot be at the centre or it would rippel
• Take cues from surroundings/literature– i.e., Rosalind’s X-ray crystallography data lying in some report
• Follow ethical guidelines
• For instance, the manner Rosalind’s X-ray crystallography data was acquired
• Best, go multidisciplinary and collaborate:
• Watson is a biologist; Crick was a physicist (so was Wilkins) and Franklin Rosalind was a chemist
• 3 fields merging toghether in the DNA research
1
,So, what is the purpose of this course?
• I would stress on dedication and creative thinking!!
• I would try to provide you with some basic concepts to help you understand molecular (neuro)pathological
concepts
• Taking cues from the surroundings/published literature, I will show you how molecular pathology is being
performed currently with a focus on molecular neuropathology
• I will help you to think comprehensively across disciplines
• Lay emphasis, where needed, on ethics on patient data and animal experimentation, and data and scientific
integrity
→prepare you for your next step in life
• Thus, molecular pathology is an advanced
understanding of the molecular
mechanisms of disease
• It bridges molecular concepts of
pathogenesis to the clinical expression of
disease in cell, tissue, and organ system
• And this is how we pathologists harness our
understanding of disease entities to develop
new diagnostics and treatments for various
human diseases
• With a focus on genotype-phenotype
correlation, it lays emphasis on the
«phenotype» and work out how genotypes
(i.e., a mutation) has effect on the
«quantity» and/or «function» of a protein
The focus here will be on dementia disorders and other neurodegenerative diseases, centered around molecular
mechanisms of cell death
don't know a lot about protein/DNA structures but more phenotypes
neurons are not allowed to die because they are post-mitotic
Molecular pathology of Human diseases
why humans? because we think we are the best species and we are selfish
we are also the best model if it comes to genotype-phenotypes correlation
• Humans have been crucial for molecular pathology studies
• We know most about genotypes of human than any other spp.
• Healthcare system (clinics) is continuously updating mutation databases
• So are researchers worldwide
– Human Mutation Database ( www.hgmd.org)
– OMIM ( www.ncbi.nlm.nih.gov)
– HUGO Mutation Database Initiative ( www.genomic.unimelb.edu.au/mdi)
We know more about phenotypes of humans than any other spp.
• Healthcare system
• Research: Any phenotype that occurs in frequency one in >109 is reported
Molecular pathology has been worked upon intensively for
– Cancer, haemoglobinopathies
– In neurodegeneration, research used to focus mainly on loss of function, but now dominant-negative and gain-of-
function effects are getting more attention
Molecular pathology studies in disease models
Hypothesis made on humans are then tested on cells and animal models: → we cannot use humans as guinnea pigs, first
in humans trials are very difficult to get
2
,Cellular models
Monolayer (2D cultures): simple, easy, low-cost.
Co-culture systems: study cell–cell interactions.
o if we want to grow neuronal cells we always use a gliacell where the neurons grow on, glia cells will not
proliferate
3D spheroids: mimic tissue architecture better than 2D.
Organoids: mini-tissues with multiple cell types, higher physiological relevance.
o we cannot put vascularisation in
iPSC-derived models: patient-specific, capture genetic background
Animal models
Natural mutations (http://www.scripps.edu/)
o website with phenotypes that are seen en frozen embryo's so you can then study what happened in the
mice and compair it with the human
Engineered mutations
o Transgenic mice – carry extra or modified genes
o Knockout (KO) mice – gene deleted, study loss-of-function → takes very long so now we usually use
CRISPR
o Knock-in mice – human mutations inserted, disease modeling
▪ knock in are more interesting because knock out you study LOF and knock in lets you understand
missense mutations and these are more common and can be LOF or GOF or dominant negative
effect
Further discussion in relevant diseases
REVISION
Molecular pathology requires more than an A type of description (see later)
3
, Ubiquitination (also called ubiquitylation) is the covalent
attachment of a small protein called ubiquitin (76 amino
acids long) to a target protein.
This attachment acts like a molecular “label” — depending
on how it’s done, it can mark the protein for:
• Degradation by the proteasome (most common),
• Altered cellular location,
• Changed activity, or
• Mediated protein–protein interactions.
The Ubiquitination Machinery
It’s a three-enzyme cascade:
1. E1: Ubiquitin-activating enzyme
o Uses ATP to activate ubiquitin and form a high-energy thioester bond with it.
o Passes ubiquitin to E2.
2. E2: Ubiquitin-conjugating enzyme
o Carries activated ubiquitin and interacts with E3.
3. E3: Ubiquitin ligase
o Recognizes the specific target protein.
o Transfers ubiquitin from E2 to a lysine residue (–NH₂ group) on the target protein.
There are only a few E1s, dozens of E2s, and hundreds of E3s — the E3 ligases give substrate specificity.
What Happens After Tagging?
• Proteasomal degradation:
The 26S proteasome recognizes K48-polyubiquitinated proteins, unfolds them, and breaks them down into
peptides.
Ubiquitin itself is recycled by deubiquitinating enzymes (DUBs).
• Non-degradative roles:
Ubiquitination can regulate signaling pathways (e.g., NF-κB activation), control DNA repair, or mediate vesicle
trafficking.
4
INHOUD
• Introduction to Molecular Pathology
• Neuropathology – Brain Structure and Function
• Tools/techniques of Molecular Pathology (limited)
• Molecular Neuropathology of:
o Alzheimer’s disease, Parkinson’s disease, Lewy body disease, Frontotemporal dementia-ALS spectrum,
Stroke, Vascular dementia, Prion diseases
o Neoplasia (limited to glioblastoma)
o Select developmental disorders/ inborn errors
PowerPoint presentations on individual assignments (10 minutes each participant) – plenty of discussion
Make an interactive presentation - 3/4/5 slides min 6min max 10 account for 6 point of the 20
If he says it’s important for the exam mark the slide!!
Have to be very active in this course
PART 1 Introduction to Molecular Pathology
What is Molecular neuropathology?
Neuropathology = Pathology of the nervous system
Molecular neuropathology = Study at a molecular (biological)
level
we mostly deal with the protein part because we are interested
in the phenotype
this tells you how the field have not been collaborating too quickly because people are working on different things
2 fields have continued, the chromosome field has stopped quickly
what is the big deal about the DNA structure? you engaged public funding and research took a big jump from there on
So, what do we learn from Watson & Crick discovery?
• Dedication, dynamism, and creative thinking are critical in any discovery!
• But never undervalue basic concepts
• First Watson/Crick model had the phosphate backbones at the center (Linus Pauling also made the same
mistake) → P backbone cannot be at the centre or it would rippel
• Take cues from surroundings/literature– i.e., Rosalind’s X-ray crystallography data lying in some report
• Follow ethical guidelines
• For instance, the manner Rosalind’s X-ray crystallography data was acquired
• Best, go multidisciplinary and collaborate:
• Watson is a biologist; Crick was a physicist (so was Wilkins) and Franklin Rosalind was a chemist
• 3 fields merging toghether in the DNA research
1
,So, what is the purpose of this course?
• I would stress on dedication and creative thinking!!
• I would try to provide you with some basic concepts to help you understand molecular (neuro)pathological
concepts
• Taking cues from the surroundings/published literature, I will show you how molecular pathology is being
performed currently with a focus on molecular neuropathology
• I will help you to think comprehensively across disciplines
• Lay emphasis, where needed, on ethics on patient data and animal experimentation, and data and scientific
integrity
→prepare you for your next step in life
• Thus, molecular pathology is an advanced
understanding of the molecular
mechanisms of disease
• It bridges molecular concepts of
pathogenesis to the clinical expression of
disease in cell, tissue, and organ system
• And this is how we pathologists harness our
understanding of disease entities to develop
new diagnostics and treatments for various
human diseases
• With a focus on genotype-phenotype
correlation, it lays emphasis on the
«phenotype» and work out how genotypes
(i.e., a mutation) has effect on the
«quantity» and/or «function» of a protein
The focus here will be on dementia disorders and other neurodegenerative diseases, centered around molecular
mechanisms of cell death
don't know a lot about protein/DNA structures but more phenotypes
neurons are not allowed to die because they are post-mitotic
Molecular pathology of Human diseases
why humans? because we think we are the best species and we are selfish
we are also the best model if it comes to genotype-phenotypes correlation
• Humans have been crucial for molecular pathology studies
• We know most about genotypes of human than any other spp.
• Healthcare system (clinics) is continuously updating mutation databases
• So are researchers worldwide
– Human Mutation Database ( www.hgmd.org)
– OMIM ( www.ncbi.nlm.nih.gov)
– HUGO Mutation Database Initiative ( www.genomic.unimelb.edu.au/mdi)
We know more about phenotypes of humans than any other spp.
• Healthcare system
• Research: Any phenotype that occurs in frequency one in >109 is reported
Molecular pathology has been worked upon intensively for
– Cancer, haemoglobinopathies
– In neurodegeneration, research used to focus mainly on loss of function, but now dominant-negative and gain-of-
function effects are getting more attention
Molecular pathology studies in disease models
Hypothesis made on humans are then tested on cells and animal models: → we cannot use humans as guinnea pigs, first
in humans trials are very difficult to get
2
,Cellular models
Monolayer (2D cultures): simple, easy, low-cost.
Co-culture systems: study cell–cell interactions.
o if we want to grow neuronal cells we always use a gliacell where the neurons grow on, glia cells will not
proliferate
3D spheroids: mimic tissue architecture better than 2D.
Organoids: mini-tissues with multiple cell types, higher physiological relevance.
o we cannot put vascularisation in
iPSC-derived models: patient-specific, capture genetic background
Animal models
Natural mutations (http://www.scripps.edu/)
o website with phenotypes that are seen en frozen embryo's so you can then study what happened in the
mice and compair it with the human
Engineered mutations
o Transgenic mice – carry extra or modified genes
o Knockout (KO) mice – gene deleted, study loss-of-function → takes very long so now we usually use
CRISPR
o Knock-in mice – human mutations inserted, disease modeling
▪ knock in are more interesting because knock out you study LOF and knock in lets you understand
missense mutations and these are more common and can be LOF or GOF or dominant negative
effect
Further discussion in relevant diseases
REVISION
Molecular pathology requires more than an A type of description (see later)
3
, Ubiquitination (also called ubiquitylation) is the covalent
attachment of a small protein called ubiquitin (76 amino
acids long) to a target protein.
This attachment acts like a molecular “label” — depending
on how it’s done, it can mark the protein for:
• Degradation by the proteasome (most common),
• Altered cellular location,
• Changed activity, or
• Mediated protein–protein interactions.
The Ubiquitination Machinery
It’s a three-enzyme cascade:
1. E1: Ubiquitin-activating enzyme
o Uses ATP to activate ubiquitin and form a high-energy thioester bond with it.
o Passes ubiquitin to E2.
2. E2: Ubiquitin-conjugating enzyme
o Carries activated ubiquitin and interacts with E3.
3. E3: Ubiquitin ligase
o Recognizes the specific target protein.
o Transfers ubiquitin from E2 to a lysine residue (–NH₂ group) on the target protein.
There are only a few E1s, dozens of E2s, and hundreds of E3s — the E3 ligases give substrate specificity.
What Happens After Tagging?
• Proteasomal degradation:
The 26S proteasome recognizes K48-polyubiquitinated proteins, unfolds them, and breaks them down into
peptides.
Ubiquitin itself is recycled by deubiquitinating enzymes (DUBs).
• Non-degradative roles:
Ubiquitination can regulate signaling pathways (e.g., NF-κB activation), control DNA repair, or mediate vesicle
trafficking.
4
