Tomas Björklund

• Professor, PhD in Neuroscience, Master of Medicine
• Professor at Lund University

Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson’s disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still u...

Synucleinopathies, characterized by intracellular aggregation of α-synuclein protein, share a number of features in pathology and disease progression. However, the vulnerable cell population differs significantly between the disorders, despite being caused by the same protein. While the vulnerability of dopamine cells in the substantia nigra to α-s...

Preclinical efficacy of continuous delivery of 3,4-dihydroxyphenylalanine (DOPA) with adeno-associated viral (AAV) vectors has recently been documented in animal models of Parkinson's disease (PD). So far, all studies have utilized a mix of two monocistronic vectors expressing either of the two genes, tyrosine hydroxylase (TH) and GTP cyclohydrolas...

Viral vector-mediated gene transfer utilizing adeno-associated viral vectors has recently entered clinical testing as a novel tool for delivery of therapeutic agents to the brain. Clinical trials in Parkinson's disease using adeno-associated viral vector-based gene therapy have shown the safety of the approach. Further efforts in this area will sho...

In Huntington's disease (HD), the mutant huntingtin protein is ubiquitously expressed. The disease was considered to be limited to the basal ganglia, but recent studies have suggested a more widespread pathology involving hypothalamic dysfunction. Here we tested the hypothesis that expression of mutant huntingtin in the hypothalamus causes metaboli...

Dopaminergic (DA) neurons exhibit significant diversity characterized by differences in morphology, anatomical location, axonal projection pattern, and selective vulnerability to disease. More recently, scRNAseq has been used to map DA neuron diversity at the level of gene expression. These studies have revealed a higher than expected molecular div...

Stem cell therapies for Parkinson’s disease are at an exciting time of development, and several clinical trials have recently been initiated. Human pluripotent stem cells are differentiated into transplantable dopamine (DA) progenitors which are proliferative at the time of grafting and undergo terminal differentiation and maturation in vivo. While...

Direct neural conversion of endogenous non-neuronal cells, such as resident glia, into therapeutic neurons has emerged as a promising strategy for brain repair, aiming to restore lost or damaged neurons. Proof-of-concept has been obtained from animal studies, yet these models do not efficiently recapitulate the complexity of the human brain, and fu...

The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a...

Cell therapy for Parkinson's disease has experienced substantial growth in the past decades with several ongoing clinical trials. Despite increasing refinement of differentiation protocols and standardization of the transplanted neural precursors, the transcriptomic analysis of cells in the transplant after its full maturation in vivo has not been...

Huntington’s disease is a fatal neurodegenerative disorder caused by an expanded CAG triplet repeat in the huntingtin (HTT) gene. Previous research focused on neuropathology in the striatum and its association with a typical movement disorder. Direct effects of mutant HTT (mHTT) in the striatum may cause neuropathology, although non-cell autonomous...

Transplantation in Parkinson's disease using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons is a promising future treatment option. However, many of the mechanisms that govern their differentiation, maturation, and integration into the host circuitry remain elusive. Here, we engrafted hESCs differentiated toward a ventral midbra...

Recent technological and conceptual advances have resulted in a plethora of exciting novel engineered adeno associated viral (AAV) vector variants. They all have unique characteristics and abilities. This review summarizes the development and their potential in treating Parkinson’s disease (PD). Clinical trials in PD have shown over the last decade...

Aggregation of α-synuclein is associated with neurodegeneration and a hallmark pathology in synucleinopathies. These aggregates are thought to function as prion-like particles where the conformation of misfolded α-synuclein determines the traits of the induced pathology, similar to prion diseases. Still, little is known about the molecular targets...

Recent advances in cell reprogramming have made it possible to form new therapeutic cells within the body itself via a process called direct conversion or lineage reprogramming. A series of studies have shown that it is possible to reprogram resident glia into new neurons within the brain parenchyma. These studies opened up for the targeted attempt...

Aggregation of α-synuclein is associated with neurodegeneration and a hallmark pathology in synucleinopathies. These aggregates are thought to function as prion-like particles where the conformation of misfolded α-synuclein determines the induced pathologys traits similar to prion diseases. Still, little is known about the molecular targets facilit...

Recombinant adeno-associated virus (rAAV) is a mammalian virus that has been altered to be used as a gene delivery vehicle. Several changes to the viral genome have made them replication deficient so that this aspect of the viral infection cycle is under full control of the experimenter, while maintaining gene expression machinery. Over the last de...

Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid stru...

Background: Cortical α-synuclein pathology plays a role in the development of cognitive dysfunction in both Parkinson's disease and dementia with Lewy bodies, although the causative cellular lesions have remained unclear. We aimed to address causal links between α-synuclein-driven pathology in the cerebral cortex and the development of cognitive i...

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These...

Chemogenetics is the process of genetically expressing a macromolecule receptor capable of modulating the activity of the cell in response to selective chemical ligand. This chapter will cover the chemogenetic technologies that are available to date, focusing on the commonly available engineered or otherwise modified ligand-gated ion channels and G...

In vivo gene therapy for neurodegenerative disorders has turned out to be a formidable challenge. It is a field not much older than twenty years, but we were many who would have predicted a much easier path towards the clinic using this treatment modality. For Parkinson’s disease patients, this has meant a frustrating wait, seeing many promising th...

Background Hypothalamus with its neuroendocrine circuitry is a main regulator of metabolism, emotional control and the reward system. It is also an important region of pathology in clinical Huntington’s disease (HD) with loss of orexin neurons. Given that the hypothalamus is affected already in prodromal HD, pathology in this area may mediate early...

Engineering of Adeno-associated viral (AAV) vector capsids through directed evolution has been used to generate novel capsids with altered tropism and function. This approach, however, involves a selection process that requires multiple generations of screenings to identify real functional capsids. Due to the random nature of this process, it is al...

Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated cli...

Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease depen...

The intricate balance between dopaminergic and cholinergic neurotransmission in the striatum has been thoroughly difficult to characterize. It was initially described as a seesaw with a competing function of dopamine versus acetylcholine. Recent technical advances however, have brought this view into question suggesting that the two systems work ra...

Detailed characterization and mapping of oligonucleotide function in vivo is generally a very time consuming effort that only allows for hypothesis driven subsampling of the full sequence to be analysed. Recent advances in deep sequencing together with highly efficient parallel oligonucleotide synthesis and cloning techniques have, however, opened...

Motor symptoms in Parkinson’s disease are attributed to degeneration of midbrain dopaminergic neurons (DNs). Heterozygosity for Engrailed-1 (En1), one of the key factors for programming and maintenance of DNs, results in a parkinsonian phenotype featuring progressive degeneration of DNs in substantia nigra pars compacta (SNpc), decreased striatal d...

Hematopoietic stem cells (HSCs) undergo a functional switch in neonatal mice hallmarked by a decrease in self-renewing divisions and entry into quiescence. Here, we investigated whether the developmental attenuation of B-1a cell output is a consequence of a shift in stem cell state during ontogeny. Using cellular barcoding for in vivo single-cell f...

Relates to Figures 2, 5 and S6. This spreadsheet contains raw values from the electrochemical recordings for each animal with calculation of peak amplitude (Amp), the rise time (Trise), and re-uptake times.

Movie S1. Representative Video Recording of Abnormal Involuntary Movements Induced through Selective Increase of cAMP in the Fetal DA Transplant Relates to Figure 4. The movie shows one representative animal from the hM3Dq + rM3Ds DREADD group. The movies are recorded 24 weeks following fetal grafting (15 weeks post-AAV injection). On 2 consecutiv...

Capsid modification is a useful strategy to create adeno-associated virus (AAV) vectors with subtype specific neuronal targeting and enhanced retrograde transport. Incorporating known cell-specific binding ligands is a rational method, but the creation of vectors without prior knowledge has the potential to reveal novel targets. Directed evolution...

Recent clinical trials have demonstrated safety and efficacy of adeno-associated virus (AAV)-mediated gene therapy, and precipitated the approval of the first gene therapy product for commercial use. Although AAV is the most commonly used vector, there are limitations due to its restricted biological activity. This can be partially overcome by alig...

In this chapter, we will cover the available design choices for enabling expression of two functional protein or RNA sequences from a single viral vector. Such vectors are very useful in the neuroscience-related field of neuronal control and modulation, e.g. using optogenetics or DREADDs, but are also desirable in applications of CRISPR/Cas9 in sit...

Recent technical advances have opened up new avenues in molecular science with great impact on both in vitro and in vivo applications. Next generation sequencing has become available to a greater extent and advanced molecular techniques and methods within RNA/DNA-editing, barcoding and generation of high diversity DNA libraries are constantly evolv...

Over the past decades, cell replacement therapy has emerged as a possible therapeutic alternative for the treatment of Parkinson's disease. Although early trials showed great promise, later double blinded trials displayed varied clinical benefits. In order to improve therapeutic potential, we combined dopaminergic cell replacement therapy with desi...

The cellular hallmarks of Parkinson's disease (PD) are the loss of nigral dopaminergic neurons and the formation of α-synuclein-enriched Lewy bodies and Lewy neurites in the remaining neurons. Based on the topographic distribution of Lewy bodies established after autopsy of brains from PD patients, Braak and coworkers hypothesized that Lewy patholo...

We used a single adeno-associated viral (AAV) vector co-expressing tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (GCH1) to investigate the relationship between vector dose, and the magnitude and rate of recovery in hemi-parkinsonian rats. Intrastriatal injections of >1E10 genomic copies (gc) of TH-GCH1 vector resulted in complete recovery in d...

Impairments in the capacity of dopaminergic neurons to handle cytoplasmic dopamine may be a critical factor underlying the selective vulnerability of midbrain dopamine neurons in Parkinson's disease. Furthermore, toxicity of α-synuclein in dopaminergic neurons has been suggested to be mediated by direct interaction between dopamine and α-synuclein...

Neurological diseases are among the most interesting applications for gene therapy. After decades of preclinical development, viral vector–based gene transfer has now reached the clinic, and early-stage clinical trials are currently in progress in three areas: congenital blindness and retinal

Dopamine replacement for Parkinson's disease (PD) have seen three major iterations of improvements since the introduction of l-3,4-dihydroxyphenylalanine (l-DOPA) pharmacotherapy: dopamine receptor agonists, ex vivo gene transfer for cell transplantation and most recently in vivo gene therapy. In this chapter, we describe the principles behind vira...

The ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. We have developed a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of the Escherichia coli dihydrofolate reductase (ecDHFR) w...

Background Non-motor features of Huntington's disease (HD) include metabolic changes such as increased appetite, insulin resistance and changes in body weight. As mutant huntingtin (htt) is ubiquitously expressed in HD as well as in most animal models that recapitulate these features, it has not been possible to determine the underlying pathology....

The once fantastic theoretical concept that patients with Parkinson's disease (PD) would receive gene therapy in an attempt to alleviate their symptoms and potentially modify the course of their disease has become a reality. On the basis of positive preclinical data, four different gene therapy approaches are currently in Phase I or Phase II clinic...

Novel therapeutic intervention based on gene therapy has moved the field of Parkinson's disease (PD) research forward during the last decade. The process of supplementing cells with genes that promote normal, healthy function promises to be an efficient way of treating diseases like PD, above and beyond what it has been possible to achieve with tra...

The introduction of L-dopa (L-3,4-dihydroxyphenylalanine) therapy 40 years ago was a revolution in the treatment of patients with Parkinson s disease (PD). With time, however, the shortcomings of oral L-dopa medication became apparent, in particular the appearance of troublesome side effects, expressed as involuntary movements (dyskinesias) that de...

Viral vector-mediated gene transfer is emerging as a novel therapeutic approach with clinical utility in treatment of Parkinson's disease. Recombinant adeno-associated viral (rAAV) vector in particular has been utilized for continuous l-3,4 dihydroxyphenylalanine (DOPA) delivery by expressing the tyrosine hydroxylase (TH) and GTP cyclohydrolase 1 (...

We examined the transduction efficiency of different adeno-associated virus (AAV) capsid serotypes encoding for green fluorescent protein (GFP) flanked by AAV2 inverted terminal repeats in the nonhuman primate basal ganglia as a prelude to translational studies, as well as clinical trials in patients with Parkinson's disease (PD). Six intact young...

Short-hairpin RNA (shRNA)-mediated gene knockdown is a powerful tool for targeted gene silencing and an emerging novel therapeutic strategy. Recent publications, however, reported unexpected toxicity after utilizing viral-mediated shRNA knockdown in vivo. Thus, it is currently unclear whether shRNA-mediated knockdown strategy can be used as a safe...

In vivo gene transfer using viral vectors is an emerging therapy for neurodegenerative diseases with a clinical impact recently demonstrated in Parkinson's disease patients. Recombinant adeno-associated viral (rAAV) vectors, in particular, provide an excellent tool for long-term expression of therapeutic genes in the brain. Here we used the [(11)C]...

The ever-evolving understanding of the neuronal systems involved in Parkinson's disease together with the recent advances in recombinant viral vector technology has led to the development of several gene therapy applications that are now entering into clinical testing phase. To date, four fundamentally different approaches have been pursued utilizi...

The introduction of L-dopa (L-3,4-dihydroxyphenylalanine) therapy 40 years ago was a revolution in the treatment of patients with Parkinson s disease (PD). With time, however, the shortcomings of oral L-dopa medication became apparent, in particular the appearance of troublesome side effects, expressed as involuntary movements (dyskinesias) that de...

Recombinant lentiviral vectors (rLV) are powerful tools for gene transfer to the central nervous system (CNS) and hold great potential as a therapeutic gene therapy strategy for neurological disorders. Recent data indicate that rLVs are suitable for functional studies in the CNS by over expression or knock down of specific proteins. Based on a vari...

The experimental field of restorative neurology continues to advance with implantation of cells or transfer of genes to treat patients with neurological disease. Both strategies have generated a consensus that demonstrates their capacity for structural and molecular brain modification in the adult brain. However, both approaches have yet to success...

During the last decade, identification of the genes involved in familial forms of Parkinson's disease (PD) has advanced our understanding of the mechanisms underlying the development of different aspects of PD. However the available animal models still remain as the main limiting factor for the development of neuroprotective therapies that can halt...

Parkinson's disease is the second most common neurodegenerative disease. It is charaterized by a progressive loss of dopamine (DA) producing neurons in the midbrain, which result in a decline of DA innervations present in the forebrain, in particular, the striatum. The disease leads to appearance of motor symptoms involving akinesia/bradykinesia, g...

Overexpression of human alpha-synuclein (alpha-syn) using recombinant adeno-associated viral (rAAV) vectors provides a novel tool to study neurodegenerative processes seen in Parkinson's disease and other synucleinopathies. We used a pseudotyped rAAV2/5 vector to express human wild-type (wt) alpha-syn, A53T mutated alpha-syn, or the green fluoresce...

Hippocampal decrease in size in response to posttraumatic stress disorder (PTSD) is still a subject of controversy. The aims of this study were to: (1) confirm previous hippocampus findings in PTSD patients compared to controls, using ethnically similar study groups where alcohol and drug abuse were non-existent; (2) test influence of disease durat...

Fetal cell transplantation for the treatment of Parkinson's and Huntington's diseases has been developed over the past two decades and is now in early clinical testing phase. Direct assessment of the graft's survival, integration into the host brain and impact on neuronal functions requires advanced in vivo neuroimaging techniques. Owing to its hig...