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Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative syndrome characterized by progressive degeneration of motor neurons that result in muscle wasting. The symptoms advance gradually to paralysis and eventually death. Most patients suffer from sporadic ALS (sALS) but 10% report a familial predisposition. Mutations in the gene encoding super­oxide dismutase-1 (SOD1) were the first identified cause of ALS. The disease mecha­nism is debated but there is a consensus that mutations in this protein confer a cytotoxic gain of function. SOD1 aggregates in motor neurons are hallmarks of ALS both in patients and in transgenic mouse models expressing a mutated form of human SOD1 (hSOD1). Recently, our group showed that SOD1 aggregates are present also in sALS patients, thus indicating a broader involvement of this protein in ALS. Misfolding and aggregation of SOD1 are dif­ficult to study in vivo since aggregate concentration in the central nervous system (CNS) is exceedingly low. The aim of this thesis was to find a method circumventing this problem to investigate the hSOD1 aggregate structure, distribution and spread in ALS disease.

Many studies provide circumstantial evidence that the wild-type hSOD1 protein can be neurotoxic. We developed the first homozygous mouse model that highly overexpresses the wild-type enzyme. These mice developed an ALS-like syndrome and become terminally ill after around 370 days. Motor neuron loss and SOD1 aggregate accumulation in the CNS were observed. This lends further support to the hypothesis of a more general involve­ment of SOD1 in human disease.

A panel of polyclonal antibodies covering 90% of the SOD1 protein was developed by our laboratory. These antibodies were shown to be highly specific for misfolded SOD1. Aggre­gated hSOD1 was purified from the CNS of terminally ill hSOD1 mice. Disordered segments in aggregated hSOD1 could be identified with these antibodies. Two aggregate strains with different structural architectures, molecular properties, and growth kinetics, were found using this novel method. The strains, denoted A and B, were also associated with different disease progression. Aggregates formed in vitro were structurally different from these strains. The results gave rise to questions about aggregate development and possible prion-like spread. To investigate this, inoculations of purified strain A and B hSOD1 seeds was performed in lumbar spinal cords of 100-day old mice carrying a hSOD1^G85R mutation. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill 200 days earlier than mice inoculated with control preparation. Interestingly, a tem­plated spread of aggregates along the neuraxis was concomitantly observed, with strain A and B provoking the buildup of their respective hSOD1 aggregate structure. The phenotypes initiated by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. To further establish the importance of hSOD1 aggregates in human disease, purification and inoculation of aggregate seeds from spinal cords of ALS patients and mice carrying the hSOD1^G127X mutation were performed. Inoculation of both human and mouse seeds as described above, induced strain A aggregation and premature fatal ALS-like disease.

In conclusion, the data presented in this thesis provide a new, straightforward method for characterization of aggregate strains in ALS, and plausibly also in other neurodegen­erative diseases. Two different prion strains of hSOD1 aggregates were identified in mice that resulted in ALS-like disease. Emerging data suggest that prion-like growth and spread of hSOD1 aggregation could be the primary pathogenic mechanism not only in hSOD1 transgenic models, but also in human ALS.

Amyotrophic lateral sclerosis (ALS) is an adult-onset fatal neurodegenerative disease characterized by a progressive degeneration of the upper and lower motor neurons. The resulting paresis begins focally, usually in one muscle, and spreads contiguously, leading to muscle wasting, progressive paralysis and eventually death. 90% of all ALS cases are sporadic, with no genetic background (sALS), while 10% are hereditary or familial (fALS). The first identified cause of ALS was mutations in the gene encoding the enzyme superoxide dismutase 1 (SOD1), which are found in 3-6% of the ALS patients. Mutations in SOD1 confer a cytotoxic gain of function on the enzyme. Cytosolic inclusions containing aggregated SOD1 in motor neurons are a hallmark of ALS, both in patients and transgenic (Tg) mice carrying mutant human SOD1s (hSOD1). These inclusions have also been reported in sporadic and familial ALS cases without SOD1 mutations, suggesting a broader role of this protein in the ALS pathology. However, the mechanism of SOD1 misfolding and aggregation, and their contribution to the disease pathogenesis, is unclear.

Our research group has recently identified two structurally different strains of hSOD1 aggregates (denoted A and B) in the central nervous system of Tg murine models expressing full-length hSOD1 variants.

The aim of this thesis is to investigate if the SOD1 aggregation is a collateral byproduct in the process of the disease, or if it drives ALS pathogenesis. In addition, this work investigates the spreading characteristic of the disease in vivo.

Human SOD1 A and B seeds were prepared from spinal cords of terminally ill hSOD1 Tg mice by ultracentrifugation through a density gradient. Minute amounts of the aggregate seeds were micro-inoculated into the lumbar spinal cord of asymptomatic recipient Tg mice, overexpressing G85R mutant hSOD1 (hSOD1^G85R). Mice inoculated with A or B aggregates developed early-onset fatal ALS-like disease, becoming terminally ill around 100 days after inoculation. This is nearly 200 days earlier than hSOD1^G85R Tg mice inoculated with a control preparation or non-inoculated mice. Concomitantly, exponentially growing templated hSOD1 aggregation developed in the recipient mice, spreading all along the neuraxis. The pathology provoked by the A and B strains differed in aggregation growth rates, disease progression rates, aggregate distribution along the neuraxis, rates of weight loss, end-stage amounts of aggregates, and histopathology.

Next, we explored the existence of mutant hSOD1 aggregates with prion-like properties in the spinal cord of ALS patients.  To this end, aggregate seeds were prepared from the spinal cord of the autopsy material of an ALS patient carrying the hSOD1^G127X truncation mutation, as well as from mice transgenic for the same mutation. The aggregates showed a strain A-like core structure. Inoculation of both the murine and human derived seeds into the lumbar spinal cord of hSOD1 expressing mice efficiently transmitted strain A aggregation, propagating rostrally throughout the neuraxis and causing premature fatal ALS-like disease. The inoculation of human or murine control seeds had no effect. The potency of the ALS patient-derived seed was exceedingly high, and the disease was initiated under conditions plausible to exist also in the human motor system. These results demonstrate for the first time, the presence of hSOD1 aggregates with prion-like properties in human ALS.

We extended the exploration of hSOD1 prion mechanisms by inoculating another recipient mouse line, with wild-type-like stability and essentially normal SOD activity. Mice that are hemizygous for the hSOD1^D90A transgene insertion do not develop ALS pathology and have normal murine lifespans (>700 days). Homozygous mice develop ALS-like disease around 400 days-of-age. Interestingly, inoculations of both strain A and B seeds into the lumbar spinal cord of hemizygous hSOD1^D90A mice induced progressive hSOD1 aggregations and premature fatal ALS-like disease after around 250 and 350 days, respectively. In contrast, hemizygous hSOD1^D90A mice inoculated with a mouse control seed died from senescence-related causes at ages beyond 700 days.

Altogether, data in this thesis shows that the hSOD1 aggregate strains are ALS transmitting prions, suggesting that prion-like growth and spread of hSOD1 aggregation is the core pathogenic mechanism of SOD1-induced ALS.