Pasterkamp lab

Neural Circuit Development and Disease

Function and signaling of axon guidance cues
 

One of the most challenging problems in biology is to understand how the billions of neurons in the mammalian nervous system “wire up” to form functional neural circuits that underlie all behavior. This has been one of the most intensely studied areas of developmental neurobiology in the past, and a number of important proteins have been identified that instruct axons to project to their specific target regions (so called axon guidance proteins). Our current work focuses on how axon guidance proteins are detected by receptor proteins on growing axons, how these receptors signal and how they are regulated. Remarkably, our nervous system contains billions of connections but no more than a hundred axon guidance proteins. How can this relatively small number of proteins set up the wiring of a disproportionally large number of connections with many different characteristics such as trajectories or synaptic partners? Evidence is emerging that the effects of axon guidance proteins are diversified and spatiotemporally controlled, vastly expanding the number of guidance decisions they can mediate.
However, the molecular details of this regulation and its relevance for vertebrate axon guidance in vivo remain incompletely understood. Another aim of our work is therefore to resolve key molecular events that enable a limited number of axon guidance proteins to dictate a vast number of wiring decisions in vivo. This work is in part supported by a VICI grant from ALW-NWO and the NWO Gravitation Program BRAINSCAPES.

Novel mouse genetics tools for studying neuronal subset connectivity
 

Many brain nuclei can be further subdivided into smaller neuronal subsets with specific molecular signatures and functions. For example, the midbrain dopamine system is composed of the ventral tegmental area (VTA) and the substantia nigra (SN). Both parts have different functions and are differentially affected in disease (SN neurons die in Parkinson disease patients). However, even within the VTA and SN smaller neuronal subsets exist. We are currently setting up subtractive genetics strategies in mice to genetically label, purify, activate or ablate functionally similar subsets of neurons in the dopamine system and other parts of the nervous system such as the habenula. Using this method, we have identified many aspects of dopaminergic pathway development (e.g. rosto-caudal extension, axonal orientation, fasciculation). This new approach will help us to study novel aspects of dopaminergic pathway development such as axonal pruning, topographic bundle organization and axonal sorting. This work is in part supported by Stichting ParkinsonFonds and the NWO Gravitation Program BRAINSCAPES.

Non-coding RNAs in neural circuit development and disease
 

Non-coding RNAs are important players in neural development and brain disease. We focus on different classes of non-coding RNAs: microRNAs (miRNAs), circular RNAs (circRNAs) and transfer RNA fragments (tRFs).

MiRNAs are small non-coding RNAs (22 nt) that bind specific mRNA targets thereby regulating their translation and stability. Deregulation of miRNAs has been implicated in various brain disorders and these molecules are considered to be promising therapeutic targets in many of these disorders. Our previous work shows that miRNAs are deregulated at different levels in patients suffering from temporal lobe epilepsy (TLE) and using a multidisciplinary approach (e.g. RNA-seq, biotin-IP, in vivo Ago IPs, epilepsy models, etc.) we aim to decipher how miRNAs contribute to the process of epileptogenesis and how these effects can be reversed.

Circular RNAs are a (re)discovered class of long non-coding RNAs with a circular structure. Besides being very stable due to their circular structure, circRNAs are enriched in the brain and in specific subcellular compartments. Most studied functions include miRNA and RNA binding protein (RBP) sponging, yet, the involvement of circRNAs in different molecular processes remains to be determined. We aim to determine which circRNAs are important for axonal development and how they affect this process. Similarly, we study deregulated circRNAs in neurological diseases, such as refractory epilepsy.

Fragments derived from tRNAs are implicated in different aspects of gene regulation. One example is their miRNA-like function. We are interested in finding out how these fragments contribute to brain disease.
Further, we are constantly working on technical approaches to detect, visualize and manipulate non-coding RNAs, both in vitro and in vivo.
Our research is in part supported by the Epilepsiefonds and the Marie Curie ITN circRTrain. Furthermore, part of this work is performed in close collaboration with InteRNA Technologies.

ALS: from genetic defects to novel therapies
 

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease affecting roughly 50,000 people at any time in Europe, and causing around 10,000 deaths each year. It can occur in any individual at anytime during adulthood. ALS is characterized by progressive degeneration of motor neurons in brain and spinal cord leading to muscle weakness. Initial manifestations are weakness of limbs or weakness in the bulbar region leading to abnormalities of speech, swallowing difficulties and facial weakness. The patient progressively becomes paralyzed and dies eventually as a result of respiratory failure. There is no cure for ALS. The only available drugs are marginally effective in extending the life span of ALS patients with a few months.

Novel treatment options are needed for this disabling and fatal disease. The lack of treatment for ALS can be attributed to an absence of validated therapeutic targets reflecting an inadequate understanding of disease mechanisms. Elucidating the ALS pathogenic mechanism is therefore essential to pave the road for therapeutic interventions. Several projects in the lab are focused on unravelling the molecular mechanisms defective in familial and sporadic ALS.

An important starting point of our work are genetic defects detected in ALS patients (e.g. detected in Project MinE). Using different molecular cell biological, -omics, and biochemical approaches in different model systems (e.g. IPSC-generated neurons or organoids) we aim to understand how specific genetic defects cause ALS and on basis of this knowledge design therapeutic strategies. This work is in part supported by Stichting ALS Nederland and H2020 (E-Rare grants “INTEGRALS” and MAXOMOD”).

In addition to ALS, we also study other neurodegenerative disorders, including Alzheimer’s and Parkinson’s disease, in part with support from Stichting ParkinsonFonds and the JPND project ‘TRIAGE’. We use similar models and approaches to dissect disease mechanisms for these disorders and for designing therapeutic approaches.

IPSC and organoid models for unraveling brain disease mechanisms
 

After 50 days in vitro, brain organoids contains neuronal regions (green) originating from stem cell clusters (red) that are radially orientated.
The use and generation of induced pluripotent stem cell (iPSC)-generated cells has increased exponentially. The possibility of generating patient-derived iPSC lines followed by differentiation into desired cell types has the promise to provide new and ground-breaking insights into disease mechanisms and can facilitate the development of therapeutic approaches. We use iPSC approaches to generate neurons, (micro)glial cells and non-neural cells for multiple translational projects. For example, motor neurons and muscle cells for research into ALS. Differentiated cultures generated from control and patient iPSCs are subjected to numerous analyses, including electrophysiology (patch clamp), calcium imaging, immunocytochemistry and (single cell) RNAseq. In addition to isolated neuronal or glial cultures, organ-on-a-chip technology is used to reconstruct neural circuits like the neuromuscular junction. This specific project is supported by by Stichting ALS Nederland and performed in close collaboration with Mimetas.

Furthermore, genome editing approaches, such as CRISPR/CAS9, are used to generate isogenic controls or cell lines carrying specific mutations. iPSCs are also used to generate various types of brain organoids allowing analysis of neuronal and glial cell morphology and function in a three-dimensional environment, for example for studying ALS and Alzheimer’s disease. Analysis of neuronal morphology is facilitated by Ultramicroscope Lightsheet imaging present in our lab.

The IPSC- and organoid lab and lightsheet imaging platform is organized in a research facility named the MIND facility. This facility is financially supported by the UMC Utrecht Brain Center, UMC Utrecht and Utrecht University, and managed by the Pasterkamp lab.

 

Prof. dr. Jeroen Pasterkamp

I am a molecular neurobiologist trying to dissect the mechanisms that underlie neural circuit development and various neurological and neurodegenerative disorders using a multidisciplinary research approach ranging from in vitro human stem cell modelling and mouse genetics to advanced 3D microscopy.

 

Group members

Andreia G. Duarte – PhD student
Molecular biologist focused on understanding (non-coding RNA)-mechanisms leading to brain disease.

RNA metabolism, primary tissue culture, Epilepsy

Astrid T. van der Geest – PhD student
My research focuses on modeling cortical thinning in C9ORF72 ALS patients using brain organoids.

brain organoid culture, ALS, iPSC culture

Daniëlle N. Vonk – Technician
I am manager of the iPSC/Organoid part of the MIND-Facility.

iPSC culture, Brain organoid culture, differentiations of iPSC into different cell types.

Eljo Y. van Battum – Postdoc
I am a developmental neurobiologist with great interest in axon guidance mechanisms and neuronal network formation.

in vivo electroporation, primary tissue culture, microscopy

Laurens M. Grossouw – PhD student
I am interested in dopaminergic axons in development and disease.

Dopaminergic development, Tissue clearing, Mouse genetics

Lauri. M. Bloemenkamp – Technician
Our research focuses on the (pre)clinical development of a novel therapeutic antibody targeting C2.

iPSC culture, iPSC derived motor neurons, complement system

Lieke L. van de Haar – PhD student
I am a molecular neuroscientist interested in habenula development, with expertise in single cell transcriptomics.

single cell RNA-sequencing, in situ hybridization, 3DISCO

Marina de Wit – Technician
Epilepsy research using human, mouse and rat tissue

Western blot, in situ hybridization, Qpcr

Marleen H. van den Munkhof – PhD student
I focus on exploring the role of Semaphorin-Plexin signalling in neural circuit development.

In utero electroporation, Tissue clearing, Primary cell culture

Marloes Verkerke – PhD student
My research focusses on the effect of APOE4, the largest genetic risk factor for late onset AD, on neuron-glia interaction in iPSC models.

2D human iPSC models, Human brain organoids, Glia

Marta Cañizares Luna – PhD student
I study the role of Ataxin-2 in ALS using mouse and human models with a particular focus on microglia; I am also interested in developing an organoid system to study microglia in disease, namely ALS and AD.

Brain organoids & iPSC culture, Microglia biology, ALS and AD

Mateja Rybiczka-Tešulov – PhD student
My research focuses on understanding molecular mechanisms that regulate midbrain dopamine neuron development.

RNA biology, In utero electroporation, 2D & 3D Imaging

Nicky van Kronenburg – Technician
My aim is to help the researchers with their animal work wherever I can.

mouse breeding, mouse genetics, In utero electroporation

Özge Düdükcü – PhD student
I’m a developmental Neurobiologist and my research focuses on the interaction between GABAergic and Dopaminergic neurons during midbrain development.

Mouse genetics, single cell RNA sequencing, Imaging

Pavol Zelina – Postdoc
My project is aimed to deepen understanding of ALS pathogenesis and to develop a cellular model of the disease.

neurobiology of ALS, iPSC derived motor neurons, axon guidance

Rianne de Jongh – PhD student
My research aims to unravel the role of non-cell autonomous processes in axon degeneration in ALS.

iPSC culture, Microfluidics, ALS

Tijana Ljubikj – PhD student
My research focuses on understanding the role of microglia in C9orf72 – ALS using cerebral organoids. I also study the involvment of TDP-43 in ALS using cerebral organoids.

brain organoid culture, microscopy/live cell imaging, iPSC culture and differentiation

Vamshidhar R. Vangoor – Postdoc
I am a Molecular Neurobiologist interested in understanding the cellular mechanisms involved in neural cell development and in disease processes.

Epilepsy, Animal models of Epilepsy, Non-coding RNAs, Primary cell culture

Youri Adolfs – Technician
I am managing the micrscopes of our department, but also the Imaging part of the MIND-Facility. Besides suporting PhD students and Post docs with there imaging projects (from staining till analesis) I also suport with cloning projects and In utero electroporation.

Microscopy (Confocal, Lightsheet), Cloning, In utero electroporation

Ilia Timpanaro – PhD student
Trying to understand ALS cell by cell

single cell sequencing, epigenetics, ALS

 

Recent papers

  1. Robinson RA, Griffiths SC, van de Haar LL, Malinauskas T, van Battum EY, Zelina P, Schwab RA, Karia D, Malinauskaite L, Brignani S, van den Munkhof MH, Düdükcü Ö, De Ruiter AA, Van den Heuvel DMA, Bishop B, Elegheert J, Aricescu AR, Pasterkamp RJ, Siebold C. Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling. Cell. 2021 Mar 11:S0092-8674(21)00234-8. doi: 10.1016/j.cell.2021.02.045. Epub ahead of print. PMID: 33740419.
  2. Brignani S, Raj DDA, Schmidt ERE, Düdükcü Ö, Adolfs Y, De Ruiter AA, Rybiczka-Tesulov M, Verhagen MG, van der Meer C, Broekhoven MH, Moreno-Bravo JA, Grossouw LM, Dumontier E, Cloutier JF, Chédotal A, Pasterkamp RJ. Remotely Produced and Axon-Derived Netrin-1 Instructs GABAergic Neuron Migration and Dopaminergic Substantia Nigra Development. Neuron. 2020 Aug 19;107(4):684-702.e9. doi: 10.1016/j.neuron.2020.05.037. Epub 2020 Jun 19. PMID: 32562661.
  3. Vangoor VR, Reschke CR, Senthilkumar K, van de Haar LL, de Wit M, Giuliani G, Broekhoven MH, Morris G, Engel T, Brennan GP, Conroy RM, van Rijen PC, Gosselaar PH, Schorge S, Schaapveld RQJ, Henshall DC, De Graan PNE, Pasterkamp RJ. Antagonizing Increased miR-135a Levels at the Chronic Stage of Experimental TLE Reduces Spontaneous Recurrent Seizures. J Neurosci. 2019 Jun 26;39(26):5064-5079. doi: 10.1523/JNEUROSCI.3014-18.2019. Epub 2019 Apr 23. PMID: 31015341; PMCID: PMC6595945.
  4. Ormel PR, Vieira de Sá R, van Bodegraven EJ, Karst H, Harschnitz O, Sneeboer MAM, Johansen LE, van Dijk RE, Scheefhals N, Berdenis van Berlekom A, Ribes Martínez E, Kling S, MacGillavry HD, van den Berg LH, Kahn RS, Hol EM, de Witte LD, Pasterkamp RJ. Microglia innately develop within cerebral organoids. Nat Commun. 2018 Oct 9;9(1):4167. doi: 10.1038/s41467-018-06684-2. PMID: 30301888; PMCID: PMC6177485.
  5. de Jongh R, Spijkers XM, Pasteuning-Vuhman S, Vulto P, Pasterkamp RJ. Neuromuscular junction-on-a-chip: ALS disease modeling and read-out development in microfluidic devices. J Neurochem. 2020 Dec 31. doi: 10.1111/jnc.15289. Epub ahead of print. PMID: 33382092.
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