Neurodegeneration and stem cells

Question 1

Aguayo et al 1982

Answer 1

Segment of sciatic nerve inserted close to cerebral cortex - CNS axons labelled close to the graft elongated in a remarkable fashion - glial environment characteristic of the PNS responsible for this

Confounded by the injury process inserted the graft into the brain - perform on a sham control

Question 2

Davies et al 1999

Answer 2

Immunohistochemistry revealed significant ECM abundance around astro-glial scars - large amounts of the chondroitin sulfate proteoglycans CS-PGs

areas of high CS-PG concentration were areas most lacking axon regeneration first notion that CS-PG is inhibitory to neurite outgrowth.

Enzyme chondroitinase ABC, cleave CS-PG GAG side chains - reverse the inhibitory effect the ECM component can have in vivo

Question 3

Bradbury et al 2002

Answer 3

ChABC treatment restored post-synaptic activity and promoted functional recovery of locomotor and proprioceptive behaviours in the lesioned rat dorsal column

Electrophysiological parameter of recovery is just one way of measuring - anatomical regeneration was limited, potentially due to other inhibitory properties of the glial scar impairing significant regrowth.

Study was limited by not publishing long term data - It would be important to investigate the influence of other inhibitory factors released by glial scars over time, (i.e Nogo and Myelin associated glycoprotein), which may have offset the benefits that ChABC has on creating a growth suiting environment.

Question 4

Desestret et al 2013

Answer 4

Demonstrated that M2 macrophage administration at subacute stages after middle cerebral occlusion (MCAO) failed to improve stroke outcomes after 2 weeks

Although this study is inconclusive due to the lack of a dose-response curve and long-term endpoints, it raises several important concerns about M2 microglia/macrophage therapy.

What is the optimal route of administration? When should the cells be administered? Is CNS penetration essential for therapeutic effects?

Question 5

Mikita et al 2011

Answer 5

EAE MS model

MRI macrophage tracking with USPIO nanoparticles and expression patterns of M1/M2 macrophages

M1/M2 equilibrium in blood promotes mild EAE

Imbalance towards M1 promotes relapsing EAE.

Administration of ex vivo activated M2 monocytes both suppressed ongoing severe EAE and increased immunomodulatory expression pattern in lesions

Question 6

Blesch et al 2003

Answer 6

Implanted primary fibroblasts genetically modified to secrete GDNF

Axon density increased in grafts (measured using image analysis), schwann cell marker detected, SC migrated into grafts (density 6 fold higher in grafts containing GDNF than not)

However, similar to treatment with anti-inhibitory molecules NO significant functional recovery, as axons fail to re-establish connectivity.

Instead, remerging axons were observed in greatest density in areas of high GDNF concentration, in the centre of the graft, trapping them in situ.

Question 7

Handarmin et al 2011

Answer 7

By incorporating a GF gradient within nanofibrous scaffolds, neurite outgrowth (distance elongated by axons) was raised by ~7%.

Question 8

Li et al 2005

Answer 8

Histological analysis highlighted the pathway hypothesis, channels for axon regrowth

Question 9

Cheah et al 2016

Answer 9

When alpha9beta1 tenascin binding integrin was expressed in DRG via an adenovirus vector twelve weeks after injury, axons grew from C6–7 level to above C1, covering a distance of more than 25 mm.

Behavioural and locomotor recovery was also observed in the animals suggesting not only neurite outgrowth, but reconnection.

Would be more limited in humans - due to greater distances

It is important to note however, that in the presence of inhibitory external molecules (Nogo, CSPGs) more modest effects.

YET when integrins were combined with kindlins, integrin activators, and the inhibitory effects of the environment could be diminished without direct specific targeting of inhibitory molecules.

Question 10

Cho et al 2017

Answer 10

Histone acetyltransferase (HAT) p300/CBP-associated factor (PCAF) is upregulated post injury by ERK signalling.

PCAF goes on to modify histones close to the promoters of RAGs, to upregulate these genes.

PCAF overexpression in spinal cord axons improves regeneration

Question 11

Olstorn et al 2011

Answer 11

Adult neural progenitor cells were collected from resections for epilepsy - seven days after ischemia in the rat, cells were pre-differentiated (factor treated) transplanted into hippocampus, after 4/10 weeks, brains were analysed with immunohistochemistry

Cells that were treated expressed neural markers at a much earlier stage (4 weeks post grafting). Migrated preferentially into ischemic lesion

HOWEVER – cells taken from epilepsy resections – perhaps pathological cells are not the best contributors, highlights the challenges of performing these experiments when cells are so challenging to find

Presence of cells found at 4 and 10 weeks - still limited evidence for any functional benefit to the cells

Question 12

Yang et al 2011

Answer 12

nestin-GFP reporter (so stem cells labelled with nestin express GFP)

Lack of ApoE increased proliferation of early NPCs within the DG - proliferation assay in-vivo used BrDu - label dividing cells

Perhaps lengthening of the S phase not increase in division - should investigate whether ApoE affects cell cycle duration

Resulted in depletion of the overall pool of Type 1 NPCs over time - quantified with GFP expressing cells

Question 13

Crews et al 2010

Answer 13

Increased BMP6 expression (Qrt-PCR) increased significantly in the brains of AD hippocampus in humans

Sections from the hippocampus of AD patients and nondemented controls were analyzed by immunohistochemistry with antibodies against the neuroblast marker DCX and the early NPC marker SOX2

Levels of SOX2 immunoreactivity were significantly reduced in the DG of AD hippocampus

Question 14

Sim et al 2021

Answer 14

Phenotypic screening identified OXS-N1 as potent proneurogenic small molecule

Screening of 1080 compounds - selected from a chemical library of 25,000 compounds - has resulted in the identification of 30 active compounds belonging to 5 chemical classes

OXS-N1 increases neurogenesis in the dentate gyrus and improves cognition in wild type mice

Affinity probes synthesise to assess the molecular interactions of OXS-N1 - Identified to interact with vimentin - novel target.

Question 15

Piccin et al 2014

Answer 15

In their work, NSCs were extracted from old brains and were transplanted into young brains

When placed into the younger environment, the older cells showed increased proliferation and migration, showing that behaviour is dictated by the environment (i.e elevated Wnt signalling)

Upon delivery of Wnt signalling molecules to the niche, (SB216763, a GSK-3b inhibitor, and Wnt3), NSC pools were expanded in vivo.

Question 16

Sierra et al 2015

Answer 16

Disease environments

For instance, in a mouse model of epilepsy induced by kainic acid, demonstrated accelerated depletion of NSCs in response to neuronal hyper-excitation

Induced their activation and terminal differentiation to reactive astrocytes thereby exhausting the quiescent pool to supply the hippocampus

Question 17

Kremer et al 2017

Answer 17

Unbiased proteomics screen to identify novel proteins expressed during neuronal differentiation using a human neural stem cell model,

Identified the proteoglycan Glypican-2 (Gpc2) as a putative secreted marker of immature neurons.

Exogenous Gpc2 binds to FGF2 and inhibits FGF2-induced neural progenitor cell proliferation. Gpc2 is enriched in neurogenic regions of the adult brain.

Gpc2 is detectable in adult human CSF, and first pilot experiments with a longitudinal cohort indicate a decrease over time.

Thus, Gpc2 may serve as a potential marker to monitor adult neurogenesis in both animal and human physiology and disease, warranting future studies.

BUT limited to only certain populations as SOX11 target gene - Long way to go

Question 18

Kumagai et al 2009

Answer 18

Explored the impact of transplantation of neurogenic and gliogenic populations of NSCs (clarified by neurosphere assays), rodents

SCI induced in mice and cells injected into lesion epicentre after 9 days

Histological analyses were preformed after 6 weeks

Area of SC larger in gliogenic group - prevented atrophy
Immunohistochemistry - PECAM-1-positive blood vessels were observed at the lesion site

Beneficial role of stem cells is the paracrine effect they have on creating a suited environment to regrowth and repair - not physical contribution as a neuron.

Although the transplantation of primary neurospheres had no effect on the functional recovery from contusive SCI, it may be beneficial for neurological diseases in which particular types of early projection neurons are selectively lost - much shorter distances are required

Question 19

Piccini et al 1999

Answer 19

D2 receptor occupancy measured with PET in PD patient to measure dopamine release from embryonic nigral transplants

In this patient, who had received a transplant in the right putamen 10 years earlier, grafts had restored both basal and drug-induced dopamine release to normal levels.

This was associated with sustained, marked clinical benefit and normalized levels of dopamine storage

However - inconsistent results with many patients experiencing dyskinesias

Question 20

Yang et al 2017 (cell free)

Answer 20

Therapeutically, application of miRNAs involved in the neuroremodelling process (miR-124) via modified exosomes has been shown to protect against ischemic injury and promote cortical neural progenitors to obtain neuronal identity

A Phase I/II trial begun in October 2018, testing the outcomes of MSC-generated exosome with miR-124, one month after cerebral infarction. Nevertheless, rapid clearance by the innate immune system in vivo means retaining exosomes in the lesion site for an extended period of time is not realistic. Therefore use of nanotechnology, as above, could be employed to achieve sustained exosome release.

Question 21

Preman et al 2021

Answer 21

Transplanting human induced pluripotent stem cell (hiPSC)-derived astrocyte progenitors expressing APOE ε3 (E3) or APOE ε4 (E4) into neonatal brains.

24 % of human astrocytes showing hypertrophic morphologies and thicker processes surrounding Aβ deposits, 12% showed atrophy

Proportion of human astrocytes undergoing morphological transformations lower in the chimeric AD brains than in the brains of AD patients

Perhaps poor modelling or allows us to study early stage of disease - not possible by tracking purely in-vivo

Failed to implicate importance of APOE genes with other elements of human pathophysiology - perhaps animal model does not facilitate such human specific disease mechanisms

Interesting to perform RNA sequencing analyses at single-cell resolution to dissect the cellular states of transplanted astrocyte -> not possible due to limited recovery after transplantation.

Question 22

Livesey et al 2012

Answer 22

Human iPSCs manipulated to form stem cell cortical rosettes

Combining retinoid signaling with inhibition of SMAD signaling to promote neural induction -directed differentiation of hESCs to cerebral cortex stem cells with high efficiency - tested with rt-PCR (expression) and immunofluorescence images of rosettes

Synaptogenesis occurs in vitro - visualised with super-res microscopy tested functionality by observing patch-clamp - spontaneous firing
Activity independent synaptogenesis - we know in human. Epilepsy

Question 23

Rowald et al 2022

Answer 23

Arrangement of electrodes targeting the ensemble of dorsal roots involved in leg and trunk movements

Developed a computational framework that informed the optimal arrangement of electrodes on a new paddle lead and guided its neurosurgical positioning personalized with MRI /CT

Developed software supporting the rapid configuration of activity-specific stimulation programs that reproduced the natural activation of motor neurons underlying each activity

Several successful case studies in patients regaining the ability to walk, swim, stand, cycle

However not natural movements any natural movement regained posited to be from remaining intact descending tracts that had become hypoactive through injury

Expensive as personalised therapy required

Improving the computational framework - Brain machine interfaces may improve activity-specific-stimulation programs.

Question 24

Flugel et al 2001

Answer 24

EAE

MBP-specific T cells retrovirally engineered to express the gene of green fluorescent protein

Preceding disease onset, large numbers of effector cells invade the CNS, with only negligible numbers left in the periphery.

Reactivated upon local encounter of their cognate CNS antigen

Question 25

Yednock et al 1992

Answer 25

Murine model of MS EAE monocytic cell lines from humans, rats, and mice were found to selectively bind to inflamed vessels in brain sections from animals with EAE

Human peripheral neutrophils did not bind, as expected given their lack of α4 integrin receptors.

Binding of human monocytes was nearly completely (>95%) inhibited by pretreatment of the tissue sample with a monoclonal antibody to α4 integrin.

Intraperitoneal injection of this same antibody 2 days after induction of EAE prevented the paralysis of the experimental animals and reduced the severity of paralysis in animals that did ultimately develop EAE.

Nataluzimab DMT

Question 26

Friese et al

Answer 26

Generated transgenic mice that expressed HLA-A3 and the 2D1 T-cell receptor (TCR) from human CD8+ T cells

After immunization with PLP found that a small proportion of these transgenic mice developed spontaneous EAE; however, this was suppressed when crossed with HLA-A2-expressing mice
90% reduction of CD8+ T cells

Protective HLA-A2 allele

Improve medical genetics of risk and diagnosis of MS

Question 27

Flugel et al (new animal model)

Answer 27

Generated T cells that were reactive against β-synuclein (abundant in grey matter and has previously been identified as a possible autoimmune antigen)

Used Nuclear translocation of the fluorescently labelled nuclear factor of activated T cells (NFAT) as an activation-dependent biosensor - we could confirm Tβ-syn cell activation within the meninges and deep within the grey matter

Tbsyn cells induce neurodegeneration - leaky BBB, evidence of neuronal damage, active microglia - similar cells identified in human patients

Frequency of β-synuclein-specific T cells was especially increased in patients with chronic-progressive MS, whereas the frequency of MBP-specific T cells was mainly increased in patients with relapsing-remitting MS

Question 28

Rinne et al 2015

Answer 28

Activation of microglia is thought to drive widespread neuronal damage so monitoring is important

PET radioligands can reveal the extent of microglial activation by quantifying the increased expression of the 18-kDa translocator protein

EAE Lewis rats

Reduction in microglial activation around lesion post fingolimod

Treatment effect of fingolimod can be monitored in vivo by measuring the degree of microglial activation surrounding the chronic DTH EAE lesion.

Promise for new outcome measures applicable in treatment studies

Question 29

Velasqueres et al 2014

Answer 29

Evaluated the effects of curcumin (polyphenol found in turmeric) on mouse OECs

Time lapse microscopy explant cultures of OECs from OMP-ZsGreen mice, (olfactory marker protein promoter drives expression of ZsGreen fluorescent protein)

Added curcumin low dose increased proliferation of OECs (not uniform across cells - as not in-vivo difficult to assess whether role would be significant with other factors present)

OECs were incubated with PD98059 (ERK inhibitor) and SB203580 (p38 MAP kinase inhibitor) - blocked the proliferative effect of curcumin suggesting ERK/MAP mediated

Phagocytosis assay was also conducted using heat-killed FITC labelled E. coli. OECs in curcumin media showed a dramatically higher amount of bacteria inside the bodies when compared with OECs in control media - however very few cells (not quantified)

Question 30

Belin et al 2019

Answer 30

Neuregulin 1 type III improves peripheral nerve myelination in a mouse model of congenital hypo-myelinating neuropathy

Mouse model of CHN with the targeted mutation of Mpz encoding the P0Q215X mutant protein.

Affects myelin protein expression (mRNA analysis of myelin associated genes

Bred Q215X mice with the HA-NRG1 type III transgenic mouse (overexpresses HA-NRG1 type III).

These mice have recovery of myelin thickness close to WT animals

Challenge in that overmyelination can be pathological especially in CNS (i.e implicated in schizophrenia) - regulated control of myelin required - local to periphery

Future studies should look to locally express in PNS

Question 31

Arthur-Farrai et al 2012

Answer 31

C-jun deletion from Schwann cells only leads to abnormal morphology, less growth factors and reduced axons regeneration.

Sciatic functional index drastically reduced

Question 32

Zhang et al 2002

Answer 32

Rabbit tibial nerve-repair model

Schwann cells were isolated from the excised rabbit tibial nerve by using the polylysine differential adhesion method - identified by immunocytochemical labeling

Tibial nerve defects created in 24 animals

In Group 1, the tibial nerve defect was repaired with interposition vein graft alone;
In Group 2, the nerve defect was repaired with a vein graft with intraluminal injection of Schwann-cell suspension
In Group 3, the nerve defect was repaired by autogenous nerve graft alone.

At 2 months postoperatively, electrophysiologic evaluation showed that an evoked muscle action potential was recorded for the animals in Group 2 and 3
Fast conduction speed, AP =/= effective functional recovery, For how long is improved?