TAR DNA binding protein of 43 kDa (TDP-43) has been implicated in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Pathologically misfolded and aggregated forms of TDP-43 are found in cytoplasmic inclusion bodies of affected neurons in these diseases. The mechanism by which TDP-43 misfolding causes disease is not well understood. We postulate that the aggregation process plays a major role in pathogenesis, and we hypothesize that oligonucleotide ligands of TDP-43 can stabilize the native functional state of the protein and ameliorate aggregation of this aggregation-prone protein. Using recombinant TDP-43 we were able to examine the extent to which various oligonucleotide molecules affects its aggregation in vitro. We have found that certain natural sequence and de novo designed oligonucleotides bind TDP-43 and prevent its natural tendency to aggregate. The clinical and therapeutic implications of these findings are discussed.

TAR DNA binding protein of 43 kDa (TDP-43) has been implicated in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Pathologically misfolded and aggregated forms of TDP-43 are found in cytoplasmic inclusion bodies of affected neurons in these diseases. The mechanism by which TDP-43 misfolding causes disease is not well understood. We postulate that the aggregation process plays a major role in pathogenesis, and we hypothesize that oligonucleotide ligands of TDP-43 can stabilize the native functional state of the protein and ameliorate aggregation of this aggregation-prone protein. Using recombinant TDP-43 we were able to examine the extent to which various oligonucleotide molecules affects its aggregation in vitro. We have found that certain natural sequence and de novo designed oligonucleotides bind TDP-43 and prevent its natural tendency to aggregate. The clinical and therapeutic implications of these findings are discussed.

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease causing degeneration of upper and lower motor neurons. Most ALS cases are sporadic; only 6% are associated with mutations in Cu, Zn superoxide dismutase (SOD1). It is believed, however, that sporadic and familiar forms of ALS share a common mechanism, where SOD1 plays an important role: SOD1 knockout mice do not develop ALS, whereas the overexpression of human SOD1 in mice produces ALS-like symptoms. Increasing evidence suggest that the SOD1 structure gains cytotoxic properties, but detailed description of the toxic species is missing. This thesis work is focused on understanding how structural and dynamic properties of SOD1 change along its folding free-energy landscape and indicates the structural hot-spots from where the cytotoxic species may originate. Thus, binding of the zinc controls folding, stability and turnover of SOD1: (i) miscoordination of Zn2+ by the Cu-ligands speeds up folding of the SOD1 core structure, however, it stabilizes SOD1 in a state where both active-site loops IV and VII are unfolded, (ii) coordination of Zn2+ in the Zn-site, induces the folding of loop VII and stabilizes the native and  functional fold of both active-site loops and (iii) the tremendous stability gain due to Zn-site metallation corresponds to a folded state’s lifetime of  &gt; 100 years, thus the cellular lifetime of SOD1 is likely controlled by Zn2+ release, which again is coupled to opening of active-site loops. Hence the active-site loops IV and VII stand out as critical and floppy parts of the SOD1 structure. Moreover, a number of ALS-associated mutations, benign to apo-SOD1 stability, are shown here to affect integrity of active-site loops in holo-SOD1, which, in turn, increases population of SOD1 species with these loops disorganized. Finally, the close relation between SOD1 and Zn2+ can also act in the reverse direction: a perturbed folding free-energy landscape of SOD1 can disturb Zn2+ homeostasis. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH. We find that the PrP from animals that are more susceptible to prion diseases, like hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.

Pathogenesis of prion diseases in animals is associated with the misfolding of the cellular prion protein PrPC to the infectious form, PrPSc. We hypothesized that an animal’s susceptibility to prions is correlated with the propensity of an animal’s PrPC to adopt a β-sheet, PrPSc-like, conformation. We have developed a method which uses circular dichroism (CD) to directly calculate the relative population of PrP molecules that adopt a β-sheet conformation or the ‘β-state’, as a function of denaturant concentration and pH. We find that the PrP from animals that are more susceptible to prion diseases, like hamsters and mice, adopt the β-state more readily than the PrP from rabbits. The X-ray crystal structure of rabbit PrP reveals a helix-capping motif that may lower the propensity to form the β-state. PrP in the β-state contains both monomeric and octameric β-structured species, and possesses cytotoxic properties.

The PMCA assay was optimized for adaptation to low level detection of PrPSc in hamster plasma. Evaluation of numerous key variables of the PMCA assay led to an optimized protocol capable of ~3 log10 amplification after 32 cycles (two 16 hour rounds). When commercially purchased normal hamster plasma was added to the PMCA reaction an accentuation in PrPSc amplification was observed (>6.75 log10 after 32 cycles). Only con-specific plasma appeared to enhance the conversion of PrPC to PrPSc, suggesting that a species-specific co-factor may be involved in assembly of protein aggregates. Serial PMCA in the presence of low level (10%) contiguous conspecific plasma resulted in the generation of de novo PrPSc after several rounds of PMCA. Although plasma significantly accentuated PrPSc amplification by PMCA, the formation of de novo PrPSc interfered with the ability of using the PMCA assay to detect prion infections in hamsters experimentally infected with 263K scrapie. / Physiology, Cell and Developmental Biology

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative syndrome of unknown etiology that most commonly affects people in middle and high age. The hallmark of ALS is a progressive and simultaneous loss of upper and lower motor neurons in the central nervous system that leads to a progressive muscle atrophy, paralysis and death usually by respiratory failure. ALS is not a pure motor neuronal syndrome; it extends beyond the motor system and affects extramotor areas of the brain as well. The majority of the patients suffer from a sporadic ALS disease (SALS) while in at least ten percent the disease appears in a familial form (FALS). Mutations in the gene encoding the antioxidant enzyme superoxide dismutase-1 (SOD1) are the most common cause of FALS. More than 165 SOD1 mutations have been described, and these confer the enzyme a cytotoxic gain of function. Evidence suggests that the toxicity results from structural instability which makes the mutated enzyme prone to misfold and form aggregates in the spinal cord and brain motor neurons. Recent studies indicate that the wild-type human SOD1 protein (wt-hSOD1) has the propensity to develop neurotoxic features. The aim of the present study was to investigate if wt-hSOD1 is involved in the pathogenesis of SALS and FALS patients lacking SOD1 mutations and to evaluate the neurotoxic effect of misfolded wt-hSOD1 protein in vivo by generating a transgenic wt-hSOD1 mice model. We produced specific SOD1-peptide-generated antibodies that could discriminate between the misfolded and native form of the enzyme and optimized a staining protocol for detection of misfolded wt-hSOD1 by immunohistochemistry and confocal microscopy of brain and spinal cord tissue. We discovered that aggregates of misfolded wt-hSOD1 were constitutively present in the cytoplasm of motor neurons in all investigated SALS patients and in FALS patients lacking SOD1 gene mutations. Interestingly, the misfolded wt-hSOD1 aggregates were also found in some motor neuron nuclei and in the nuclei of the surrounding glial cells, mainly astrocytes but also microglia and oligodendrocytes, indicating that misfolded wt-hSOD1 protein aggregates may exert intranuclear toxicity. We compared our findings to FALS with SOD1 mutations by investigating brain and spinal cord tissue from patients homozygous for the D90A SOD1 mutation, a common SOD1 mutation that encodes a stable SOD1 protein with a wild-type-like enzyme activity. We observed a similar morphology with a profound loss of motor neurons and aggregates of misfolded SOD1 in the remaining motor neuron. Interestingly, we found gliosis and microvacuolar degeneration in the superficial lamina of the frontal and temporal lobe, indicating a possible frontotemporal lobar dementia in addition to the ALS disorder. Our morphological and biochemical findings were tested in vivo by generating homozygous transgenic mice that over expressed wt-hSOD1. These mice developed a fatal ALS-like disease, mimicking the one seen in mice expressing mutated hSOD1. The wt-hSOD1 mice showed a slower weight gain compared to non-transgenic mice and developed a progressive ALS-like hind-leg paresis. Aggregates of misfolded wt-hSOD1 were found in the brain and spinal cord neurons similar to those in humans accompanied by a loss of 41 % of motor neurons compared to non-transgenic litter mates. In conclusion, we found misfolded wt-hSOD1 aggregates in the cytoplasm and nuclei of motor neurons and glial cells in all patients suffering from ALS syndrome. Notable is the fact that misfolded wt-hSOD1 aggregates were also detected in FALS patients lacking SOD1 mutations indicating a role for SOD1 even when other genetic mutations are present. The neurotoxicity of misfolded wt-hSOD1 protein was confirmed in vivo by wt-hSOD1 transgenic mice that developed a fatal ALS-like disease. Taken together, our results support the notion that misfolded wt-hSOD1 could be generally involved and play a decisive role in the pathogenesis of all forms of ALS.

One of the key molecular events in the transmissible spongiform encephalopathies or prion diseases is the conformational conversion of the cellular prion protein PrPC into the misfolded and pathogenic isoform, PrPSc. Prion diseases are fatal neurodegenerative conditions affecting humans and other animal species, which present with diverse clinical and neuropathological phenotypes. In humans, prion diseases can occur as sporadic, familial or acquired forms. Sporadic Creutzfeldt–Jakob disease (sCJD) accounts for the majority of cases. The current classification system of human prion diseases recognizes several distinct clinico-pathological entities including sCJD, variant Creutzfeldt-Jakob disease (vCJD), Gerstmann–Straussler–Scheinker syndrome, fatal familial insomnia and variably protease-sensitive proteinopathy. Prion protein gene (PRNP) mutations and polymorphisms, and PrPSc types have a profound effect on these clinico-pathological phenotypes. Prion diseases of sheep and goats, cattle, and cervids are all actual animal health problems and present potential risks to human health. Thus far the only known zoonotic prion disease is bovine spongiform encephalopathy, which has resulted in vCJD in humans. The recognition of new forms of prion diseases in animal and humans has generated increased awareness of the animal and public health risks associated with prion disease. However the mechanisms involved in prion replication, transmission, and neurodegeneration remain poorly understood. This thesis uses in vitro PrP conversion assays (protein misfolding cyclic amplification and real time quaking-induced conversion) to model different aspects of human prion replication: Molecular susceptibility, genetic compatibility, spontaneous formation and the effect of molecules that might enhance or prevent conversion were each investigated in order to obtain a better understanding of the molecular mechanism of the prion replication. I have addressed the hypothesis that the major determinant factors in prion disease pathogenesis (PRNP genetics, PrPSc types and species barriers) are intrinsic to the prion protein conversion process and their effects can be faithfully recapitulated by in vitro conversion assays. The results shows that in vitro conversion assays used in this thesis can model the combined effects of different PrP type and genotypes, can replicate aspects of cross-species transmission potential and provide information about molecular barrier to zoonotic transmission, can model de novo PrPSc formation, and can assess the potential impact of chaperones on conversion of the human prion protein. In summary, this work provides evidence that the origin, propagation and transmission of prions can be meaningfully investigated in cell-free systems.

High-density lipoproteins and their major protein, apolipoprotein A-I (apoA-I), remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, non-variant full-length apoA-I deposits as fibrils in arteries contributing to atherosclerosis. In hereditary amyloidosis (AApoAI), a potentially fatal disease, N-terminal fragments of variant apoA-I deposit in vital organs and damage them. There is no cure for apoA-I amyloidosis and its structural basis is unknown. Previously, AApoAI mutations were mapped on the crystal structure of the human C-terminally truncated Δ(185-243)apoA-I. The results suggested that the mutation-induced destabilization of the lipid-free protein initiates β-aggregation. Our biophysical studies showed that amyloidogenic mutations G26R, W50R, F71Y and L170P did not necessarily destabilize the native structure, prompting us to search for additional triggers of apoA-I misfolding. We mapped residue segments predicted to promote β-aggregation (termed amyloid hot spots) on the atomic structure of ∆(185-243)apoA-I. The results suggested that perturbed packing of these hot spots, particularly residues 14-22, triggers amyloidosis. This enabled us to propose the first molecular mechanism of apoA-I misfolding. To explore a potential mechanism, we combined structural, stability, dynamics and functional studies of several amyloidogenic mutants and a non-amyloidogenic control, L159R. All mutants reduced structural protection of the segment 14-22, supporting our hypothesis that increased dynamics of this segment triggers AApoAI. The non-amyloidogenic mutant showed helical unfolding near the mutation site indicating susceptibility to proteolysis. We propose that the major factors that make apoA-I amyloidogenic are reduced protection of the major amyloidogenic segments combined with the structural integrity of the four-helix bundle to facilitate protein aggregation. Together, our results suggest that the fate of apoA-I in vivo depends on the balance between its misfolding, proteolysis, and protective protein-lipid interactions. Our structural and bioinformatics analysis of other members of the apolipoprotein family (A-II, A-IV, A-V, B, C-I, C-II, C-III, E, SAA) showed that apolipoproteins’ propensity to form amyloid is rooted in the proteins’ hydrophobicity, which is key to the lipid binding ability. The overlap of functional and pathologic interfaces suggests competition between normal protein function and misfolding. Therefore, increasing apolipoprotein retention on the lipid surface provides a potential therapeutic strategy against amyloidosis.