NCL Research Newsletter October 2017

Content

Main Topic(s):

            3rd International JNCL Young Investigator Symposium

            Interview with Dr. Wishart - winner of the 7th NCL Research Award

Scientific and Medical Meetings

Recent Publications

New tools for CLN3 research

Grants, Awards and Open Positions

          Call for 8th NCL Research Award

          Call for Neurodegeneration Award 2018

          German Medical Award 2017

Miscellaneous

 

 

Main Topic(s):

3rd International JNCL Young Investigator Symposium

Young investigator symposium, Hamburg, August 6-8, 2017

 

Participants of the 3rd JNCL Young Investigator Symposium

 

Thanks to the generous financial support the NCL Foundation received from a number of companies, organizations and foundations, we were able to organize the 3rd International JNCL Young Investigator Symposium that recently took place in Hamburg from August 6-8.  Eighteen young investigators participated, all working in different laboratories that are located in seven different countries (USA, CAN, GBR, D, LUX, NLD, LBN). Each young investigator presented a short talk on his/her CLN3 and other NCL disease-related research activities thereby providing a platform for the young investigators to meet and discuss their science with colleagues. The meeting also provided a platform for the young investigators to learn more about daily life with CLN3 disease as the meeting started with a presentation by a mother of a child with CLN3 disease.

 

Our young investigators also had the opportunity to listen to and discuss with a number of highly renowned keynote speakers including scientists and clinicians that covered topics including the future of Batten and gene therapy (Dr. Myriam Mirza, Germany), a clinical overview of NCL diseases (Dr. Eva Wibbeler, UKE Hamburg, Germany), “Captain lysosome – Master of the cellular universe” (Prof. Steven Walkley, Albert Einstein Institute, New York, USA), “Accurate genetic models of CLN3 disease” (Prof. Susan Cotman, Massachusetts General Hospital, Boston, USA), “Proteomic mapping of CLN3 synaptic populations to identify novel modifiers” (Dr. Thomas Wishart, University of Edinburgh, UK), “Calcium release from lysosomes and its significance for the physiology and pathophysiology of neurons” (Prof. Nigel Emptage, University of Oxford, UK), and endolysosomal ion channels as a possible new approach to treat NCL (Dr. Christian Grimm, University of Munich, Germany). Altogether, the program included 26 lectures and the presentations by the young investigators covering a wide variety of topics such as the development of biomarkers and clinical readouts, JNCL (CLN3) disease models in zebrafish, mouse, minipig, induced pluripotent human stem cell-derived neuronal and organoid (retina, brain) models, and CLN3 models in lower organisms (yeast, fly). It also included different presentations on therapeutic approaches such as gene therapy, the use of splice-switching oligonucleotides, and the use of low molecular weight compounds targeting lysosomal ion channels.

 

One of the enigmas and central themes in CLN3 research still remains the elucidation of the function of CLN3 and Dr. Luke Haslett (University of Cardiff, UK) presented latest findings with regard to roles of CLN3 in lysosomal ion homeostasis. CLN3 function, its enigmatic role in lysosome biology, and the role of the lysosome as central coordinator of cell metabolism were recurrent themes during the meeting. A number of very interesting working hypothesis exist and were part of the topics presented. Nonetheless, the veil of mystery around CLN3 function remains to be fully lifted and we all hope that the recent progress in research will make this happen soon. The last day of the meeting included two very lively workshops on themes (CLN3 function and overlaps with other neurodegenerative diseases), selected and chaired by participants. Lively, also because of the very active participation of our young investigators in these discussions. We closed the meeting with a session in which every participant was asked for her/his impressions including feedback on the content and organization of the meeting. We also received some very good suggestions and on behalf of all the organizers, we would once more like to thank all the participants for their great contributions. The general feedback certainly was that the scope of this meeting to bring together young investigators from across the globe was excellent and therefore deserves to be organized on a more regular basis.

 

 

Interview with Dr. Wishart - winner of the 7th NCL Research Award

1. How did you both become aware of CLN3 disease?

Tom and Maica’s interests are in using differentially vulnerable neuronal populations to elucidate the mechanisms governing the stability of the nervous system and therefore identify novel factors capable of regulating neurodegenerative disease processes. Tom became aware of “lysosomal storage disorders” during his first post doctoral position when he was working for Prof. Tom Gillingwater in 2007. Maica became familiar with the disease when she undertook a masters project in Tom Wishart’s laboratory as part of a Eurolife exchange programme in 2012.

 

Dr. Tom Wishart and Maica Llavero Hurtado, winner of the 7th NCL Research Award

 

2. What made you decide to apply for the NCL Research Award? Why did you apply together?

We applied for this award because we felt that we had solid preliminary but very promising data which would be important to follow up. This kind of award is incredibly valuable to us as the work proposed would be difficult to gain conventional grant funding for (due to its short term nature), but which we expect to enable us to leverage much larger sums of longer term funding.

We applied together because Tom is Maica’s current supervisor for her PhD project, which produced the exciting data we plan to progress. We are both very enthusiastic about the potential insights that this study could have for our understanding of CLN3 (and related) conditions and the impact it could have on channeling subsequent avenues of exploration for appropriate disease modifying agents.

 

3. Your lab focuses on disease-relevant changes in the synapse. You apply proteomics to try and identify molecular changes in the hope that these will provide novel therapeutic entry points. Can you explain how this might work?

Synaptic alterations in both structure and function are an early pathological feature of a broad range of neurodegenerative conditions, regardless of if they are caused by infectious agents such as prions, result in conditions associated with advancing age such as Alzheimer’s and Parkinson’s disease, or manifest in early onset degeneration such as in spinal muscular atrophy or the lysosomal storage disorders.

Using synaptic alterations as an entry point into a conserved process we believe that by examining differentially vulnerable neuronal populations (i.e. not all brain regions are affected equally in all neurodegenerative conditions) we can tease out the molecular changes which correlate with this altered “vulnerability” and/or disease progression. By identifying such molecular candidates we can then determine if they are capable of regulating the stability of the nervous system, and/or regulating disease progression, and if they are targetable with existing pharmacological agents.

 

4. You use mainly Drosophila as a target validation model while dealing with a human disease. How do you deal with major concerns of translating findings in this or other organisms to the clinic?

We use mammalian systems for the identification of candidates to test. We couple that with assessment of the relevance of candidates to the human condition by looking at expression in human samples. We also use simple but rapid models such as Drosophila to help refine the candidate list by identifying which of those factors are actually capable of regulating the stability of the nervous system or the “phenotype” caused by a disease specific alteration. There is a relatively high degree of homology in terms of the proteins expressed in the nervous system in flies and mammals, but the key is that we would not stop there. Such screening systems are a stepping stone to help refine our candidate list i.e. they not only correlate with the pathology as identified in mammalian systems but can also actually do something to the process in a living (albeit simple) organism. Then we would seek to take successful candidates back up into higher order mammalian models. We therefore see the use of Drosophila as important refinement steps which will ultimately help to save time, potential waste of higher order animal model lives and money on the road to finding something beneficial to patients.

 

5. What other collaborative efforts would you like to see to support your project?

Rapid and simplified access to human patient samples is critical to establish the relevance of the factors we identify using model systems.

Integration with other groups using different model systems would be of great interest. The more species (within reason) in which candidates identified can have a beneficial effect, the more likely you have identified a species independent factor which will translate to human systems.

Access to drug libraries – the ability to have access to existing compounds for testing i.e re-purposing existing compounds which have perhaps failed in other systems such as cancer biology. This would (if applicable) be more efficient than new development as many characteristics of the compounds limitations, interactions and dosage effects will already likely be mapped out. Other groups would then have the ability to take successful compounds through refinement processes to make them more specific and/or increase activity etc.

Large animal models for pre-clinical studies are key – toxicity, dosage, kinetics, multi systemic physiological investigations etc. can all be carried out in a manner more akin to human trials and should therefore increase the likelihood of effective translation.

 

 

Scientific and Medical Meetings

German Academy for Rare Neurological Diseases – Symposium, Nov 29-Dez 1, 2017, Wartburg, Germany

In a new academy has been founded in Germany focussing on rare neurological disorders. It aims at neurologists, neuropaediatricians, human geneticists, biologists and neuro-researchers. For more information please consult here (in German).

 

EMBO workshop titled "Lysosomes and metabolism", May 6-9, 2018, Naples, Italy

This workshop covers recent aspects of lysosomal biology linking it to cellular and organismal metabolism as well as human metabolic diseases. The aim is to support exchange on lysosomes and metabolism and to enable fruitful discussions among young and senior scientists. For more information visit here.

 

Medical Education

German physicians can earn 2 CME-points by visiting an online education about NCL. There is an English version of the education movie. It is found here.

 

 

Recent Publications

CLN1

Profound pathology in the human spinal cord. Targeting the spinal cord via gene transfer vector (using an adenoassociated virus) prevented pathology and produced significant improvements in life span and motor function in Ppt1-/- mice.

 

Using proteomic evidence the authors suggest that PPT1 deficiency could be considered as a ciliopathy. Cell and brain preparations from Ppt1-/- mice exhibit fewer cells with cilia and abnormally longer cilia. The analysis revealed a difference in the distribution and levels of the modified proteins of cilia in the retina of mutant and WT.

 

CLN2

REVIEW about Gemifibrozil (gem) as a potential treatment for NCL: gem lowers lipids. Among 200 non-NCL children (but NCL-related problems) found in literature treated with this drug. Has potential as a non-invasive treatment.

 

Represents an initial step to develop consensus-based management guideline for CNL2 disease - maintenance: quality of life.

 

Gem stimulates lysosomal biogenesis and induces anti-inflammation. CLN2 -/-mice fed with gem lived longer and had better motor activity than control mice.

 

CLN3

Treatment with clinically approved immunomodulatory compounds (fingolimod & teriflunomide) maybe suitable to attenuate progression of orphan diseases with pathogenically relevant neuroinflammation, according to results obtained in mouse model.

 

Lysosomal enzyme cathepsin D (CTSD) mediates the proteolytic cleavage of PSAP precursor into saposins A-D. Direct involvement of CLN3 in the regulation of CTSD activity.


In the social amoeba Dictyostelium discoideum, loss of the CLN3 homolog, Cln3, reduces adhesion during early development, which delays streaming and aggregation. Cln3 localizes primarily to the contractile vacuole system and to compartments of the endocytic pathway. Cln3 co-localized with a Golgi marker. Proteome analyses were done. This study indicates that Cln3 functions in both conventional and unconventional protein secretion.

 

Generation of knock-in YFP-fusions for CLN3 and CLN7 in Drosophila, which are expressed at endogenous levels. CLN7 and CLN3 were expressed in the CNS of Drosophila larva and were predominantly expressed in the glia that form the insect blood-brain-barrier. CLN7 was also expressed in neurons in the developing visual system. CLN3 was strongly expressed in the excretory and osmoregulatory Malpighian tubules.

 

Electrophysiological recordings were performed in the CA1 hippocampus (HPC) and visual cortex (VC) of acute brain slices from Cln3deltaex7/8 mice. Field excitatory post-synaptic potential and population spike amplitudes, were not altered in Cln3deltaex7/8 CA1 and VC neurons at any age. Fiber volley amplitudes were significantly increased in Cln3deltaex7/8 neurons in the HPC at 1month as well as layer II/III of the VC at 1 and 4months, suggesting increased axonal excitability.

 

Three children with CLN3 disease underwent ophthalmological examination including visual acuity, fundus photography, fundus autofluorescence, electrophysiology, Goldmann visual fields, and SD-OCT. A predominant loss of the first and second neuron retinal layers progressing from the macula to the periphery was identifed. The retinal nerve fibre layer displayed gliosis. Functional testing by multifocal ERG reflected the degenerative progress.


Profiled and compared molecular composition of differentially affected presynaptic populations from the CLN3 KO mouse brain using proteomics. Identified molecular cascades and protein candidates. Some of these were tested in a Drosophila CLN3 model and shown to modify the CLN3-neurodegenerative phenotype in vivo.

 

DNAJC5/CSPalpha/CLN4

Demonstration that auto-fluorescent storage materials (AFSM) accumulation correlates with CSPalpha (co-chaperone protein) aggregation. CSPalpha WT, mutant and aggregated CSPalpha are degraded mainly by an autophagy-lysosome pathway (ALP), but disease exhibits a faster rate of degradation and suggests that CSPalpha can support drug screening as biomarker.


CLN5

New identified crucial role of CLN5 in neurogenesis -> neurogenesis is increased in Cln5-deficient mice, NPCs (neural stem/progenitor cells) are increased, their migration is reduced and differentiation towards the neuronal lineage is increased with functional alterations in the NPCs. Moreover, this correlates with the increased expression of proinflammatory cytokine IL-1ß.

 

Induced pluripotent stem cells (iPSCs) by reprogramming skin fibroblasts from a mutation-carrier into neuronal lineage cells - accumulation of AFSM, but in addition abnormalities in intracellular organelles were detected and aberrations in neuronal sphingolipid transportation.

 

CLN5 is initially translated as a type II transmembrane protein and is subsequently cleaved by SPPL3 (protease family) into a mature soluble consisting of residues 97-407. The remaining N-terminal fragment is then cleaved and degraded in the proteasome. Furthermore, the biology of CLN5 was characterized - hope to identify novel therapeutic strategy.


CLN6

AlphaB-crystallin (AlphaBC) is a small heat shock protein. Mutations in the AlphaBC gene are linked to Alpha-crystallinopathy, a hereditary myopathy histologically characterized by intracellular accumulation of protein aggregates. The ER transmembrane CLN6 protein was identified as a TMalphaBC's binder, probably acting as a downstream effector of TMalphaBC.

 

Progranulin / CLN11

The deficiency of progranulin (PGRN) alters in lysosome abundance and morphology in mouse neurons. (PGRN loss leads to accumulation of unsaturated triacylglycerides and enriches lysosome lipidomes) brain lipid composition in mice and humans are disease-specific if PRGN lacks. Transcriptomic analysis of PGRN-deficient mouse brains show distinct expression pattern of lysosomal and lipid metabolic genes.

 

PGRN is processed in ~10 kDa peptides intracellularly in different cell types. This processing is dependent on lysosomal activities. In vitro demonstration the involvement of multiple cathepsins in PGRN processing and cleaving.

 

Identification and characterization of antibodies that reliably detect different granulins (GRNs) by immunoblot and immunocytochemistry. Endogenous GRNs produced continuously; PGRNs rapidly are transformed into stable GRNs within lysosomes - dysfunction inhibits processing. Multiple GRNs are haploinsufficient in frontotemporal dementia (FTD)-GRN-patients.

 

Description that Loss-of-function causes FTD and NCL depending on the number of allels affected. Studying mice lacking PGRNs - discovery that deficiency leads to impairment of autophagy.

 

Prosaposin (PSAP, precursor of saposin activator) interacts with PGRN; interaction requires linker region, mutation in this region cause failure of interaction.

 

PSAP and PGRN facilitate each other’s lysosome trafficking. Mice with reduced PSAP expression showed FTLD-like pathology and behaviour.

 

Heterozygous mutations in GRN gene leads to FTD and homozygous mutation cause NCL. Haploinsufficency leads to NCL-like features in humans; noninvasive retinal imaging showed abnormalities in heterozygous GRN mutation carriers.

 

Canine neuronal ceroid lipofuscinoses:

A Dachshund model with a null mutation in one of the relevant NCL genes is utilized to investigate potential therapeutic interventions, including enzyme replacement and gene therapies.

 

Biomarkers

Aim of the work was to identify biomarkers for different NCL types; proteomic and biochemical methods were used to analyse lysosomal proteins. Protein expression of CLN2 and CLN3 were most similar. Significant alterations in protein expression were identified in each NCL using isobaric labelling/quant. mass spectrometry à additional validation is require.

 

Atp13a2 / CLN12

WT and ATP31A2-deficient mice at different age received saline or manganese (Mn) for 45 days. Mn enhanced the sensorimotor function, increased autofluorescence and increased insoluble alpha-synuclein in older ATP13A2-deficient mice. Mn concentration in mutation carrier was higher than in control - indicates ATP13A2-loss leads to increase sensitivity to Mn.

 

iPSC

Review about different strategies for reprogramming and differentiation of iPCSs. Discussion of key challenges and opportunities in the area of rare diseases.

 

New tools for CLN3 research

With support of the NCL foundation, the laboratory of Prof. Jens Schwamborn in Luxemburg has developed a first isogenic pair of human induced pluripotent stem cell (iPSC) lines containing either two copies of the wildtype CLN3 gene or two copies of the CLN3 gene with the Q352X JNCL mutation. The technology that was used to edit the CLN3 locus in the iPSC line is described in a paper accepted for publication in Stem Cell Reports. It is entitled "FACS assisted CRISPR-Cas9 genome editing facilitates Parkinson’s disease modeling" by Arias-Fuenzalida J, Jarazo J, Qing X, Walter J, Gomez-Giro G, Nickels SL, Zaehres H, Schöler HR, and Schwamborn JC. For requests to obtain and use these lines please contact Prof. Schwamborn (jens.schwamborn@uni.lu). Thanks to great efforts by the Beyond Batten Disease Foundation (BBDF) in collaboration with the New York Stem Cell Foundation (NYSCF), a great set and a still growing collection of human CLN3 iPSC lines is now made available to the research community.

 

 

Grants, Awards and Open Positions

New project on cardiac changes in CLN3 disease

June 1st was the start date of a new PhD fellowship project entitled “Cardiac changes in CLN3”. The principal investigator is Dr. Katja Rietdorf, and is performed in close collaboration with Dr. Martin D. Bootman, also at The Open University in Milton Keynes, UK. This project is a collaborative funding project supported by the NCL Foundation, the two Dutch foundations Batten Research Funds (BRF) and Beat Batten (BB), the German foundation Bijou Brigitte, and by Professor Fraser McDonald from Kings College, London. Cardiac dysfunction and pathological changes in cardiomyocytes are common co-morbidities in patients with a mutation in the CLN3 gene. To date, the cause of the cardiac defects is unknown and the aim of this project is to provide better insights into this currently rather neglected, but significant, co-morbidity observed in CLN3 patients. Understanding the mechanisms of cardiac dysfunction in NCL will hopefully help to better understand the function of CLN3, and lead to therapeutic interventions that can be applied before cardiac effects of the disease become manifest. Dr. Katja Rietdorf’s laboratory has longstanding experience working with both primary adult and neonatal cardiomyocytes, using a large variety of live-cell imaging techniques and her work identifying TPCs as the endolysosomally localised NAADP receptor (Calcraft et al., 2010) has given her experience in a wide range of endo-lysosomal trafficking assays, as well as lysosomal pH and Ca2+ measurements. Aside from her great expertise in cardiomyocyte biology, Dr. Katja Rietdorf is “borne to bike”. In 2016 she became 12 h National Champion in the UK.

 

 


Images of primary neonatal rat ventricular cardiac myocytes, stained with a fluorescent calcium indicator (Cal-520),

a lysosomal stain (LysoBrite), and a merged image illustrating that the lysosomes are found in the whole cell,

including the extensions. 

 

Funding periods of the 5th and 6th NCL Research Awards completed

Dr. Marco Sardiello (Baylor College of Medicine, Houston, Texas, USA) was the winner of the 5th NCL Research Award for his project entitled “Molecular pathogenesis of juvenile NCL”. This project was co-funded by the Beyond Batten Disease Foundation, AstraZeneca, and was also supported by funds raised at charity events. The project was conducted by Dr. Alberto di Ronza who used a proteomics-based approach to successfully define some very specific changes in the process of lysosomal trafficking in CLN3 tissues. The NCL funding period is completed and the project continues. Many tools were generated and both these tools as well as the many data generated provide a solid basis for sustainability of the project and for new grant proposals.

 

Alberto di Ronza and Frank Stehr


Dr. Christian Grimm (Ludwig-Maximilian-University, Munich, Germany) was the winner of the 6th NCL Research Award for his project entitled “Electrophysiological analysis of NCL causing CLN3 mutant proteins: a critical step towards functional understanding of Batten disease”. This project was co-sponsored by IMS-Health, the Liselotte-Paulsen Stiftung, United Charity, and supported by funds raised at charity events. The project was conducted by Dr. Cheng-Chang Chen, who continues to use innovative endolysosomal patch-clamping e.a. technologies. Accordingly, Dr. Grimm’s team has been able and continues to gain deeper insights into the role of CLN3 in lysosomes and mechanisms that might be engaged using low molecular weight compounds, to try to compensate for the loss-of-function of CLN3. The results obtained so far formed the basis for new collaborative initiatives and additional grant applications aimed at sustaining the research activities.

 


Cheng-Chang Chen and Abuzar Kaleem in Hamburg

 

 

Call for 8th NCL Research Award

Also this year the NCL Foundation announces a call for its NCL Research Award (50,000 EUR) to support an innovative pilot project at the Postdoctoral fellowship level. We highly encourage junior scientists, clinical researchers and medical fellows worldwide to submit projects that hold promise to help find and push forward therapies for CLN3 disease. We also highly encourage applicants that work in disease areas outside NCL, provided the proposed research is relevant to elucidate the role of CLN3 or find a cure for this disease.

 

You can find the application forms on this website.

 

Please, note that it is a 2-stage process. First of all, we need to receive your Letter of Intent before we accept your full application.

 

Deadline for handing in all required documents: November 30, 2017

 

We are looking forward to receiving your application via email to:  Research@ncl-foundation.com

 

Call for Neurodegeneration Award 2018

Thanks to great support from the Joachim Herz Stiftung in Hamburg, the NCL Foundation is pleased to announce a call for new proposals. The grant (100.000 EUR) covers a postdoctoral fellowship salary for two years. The overall scope of this grant is to foster and improve synergies in research on CLN3 childhood dementia and age-related neurodegeneration. Therefore, applications must meet the following requirements:

 

1. Two collaborating partners must co-apply for the fellowship

2. One partner must actively engage in CLN3 research

3. The second partner must be engaged in research on age-related neurodegeneration.

 

Ideally, postdoctoral candidates should conduct a considerable part of their work in both laboratories. Laboratories interested in applying should first send us a brief letter of intent. After acceptance you will be asked to send in a full proposal. The application form is HERE. For further informations, please click here.

 

Deadline: February 28, 2018

 

German Medical Award 2017

The NCL Foundation has won the GERMAN MEDICAL AWARD 2017 in the category “Charity”. The award ceremony will take place on November 25, 2017 in the Sheraton Grand Hotel Esplanade in Berlin. Tickets are still available. Here you can register.

For more information please visit: www.germanmedicalaward.com.

 

 

Open Position(s)

Postdoctoral position. Immediate opening for a postdoctoral fellow in the Sidney Weisner Laboratory for Genetic Neurological Disease at the Rose F. Kennedy Center and Department of Neuroscience at the Albert Einstein College of Medicine. The successful applicant will join the director of this laboratory, Dr. Steve Walkley, and a dedicated research group focused on understanding the underlying neuropathogenesis of lysosomal disorders as well as on developing new therapeutics for these disorders. If you are interested in applying, please get in touch with:

 

Dr. Steven U. Walkley

Director, Rose F. Kennedy Intellectual and Developmental Disabilities Research Center

Head, Sidney Weisner Laboratory of Genetic Neurological Disease

Departments of Neuroscience, Pathology and Neurology

Rose F. Kennedy Center, Rm 618

Albert Einstein College of Medicine

1410 Pelham Parkway South

Bronx, NY 10461 USA

718-430-4025 (telephone)

718-430-8821 (fax)

Steve.Walkley@einstein.yu.edu

 

 

Miscellaneous

Position paper on gene therapy

Recently, we wrote a position paper regarding developments in the field of gene therapy for NCL. For the moment, this document only exists in German and the target audience is mainly patients and their families. The goal of this document is first of all to explain our foundation’s clear and very supportive position with regard to moving such therapies forward to the clinic for proof-of-concept studies in patients. Gene therapy creates great expectations because it tries to tackle the roots of the problem by providing the cells of a patient with a healthy functional copy of the gene that is defect. At the same time, risks, including risks of failure, should not be ignored. Also, expectations have to be realistic and need to be well managed, explained and understood. Patients and their families must feel confident when taking a decision to yes/no take part in future clinical trials. A main leg of our foundation is facilitating research and we feel it is our duty to offer support to patients and their families, to help them understand the research steps that are needed to successfully move gene therapy to the clinic and the many open questions that remain as we travel this route. Therefore, we do provide independent and objective views on progress that is being made and remaining open questions related to translating NCL gene therapy to the clinic.