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The findings that amyotrophic lateral sclerosis (ALS) patients almost universally display pathological mislocalization of the RNA-binding protein TDP-43 and that mutations in its gene cause familial ALS have nominated altered RNA metabolism as a disease mechanism. However, the RNAs regulated by TDP-43 in motor neurons and their connection to neuropathy remain to be identified. Here we report transcripts whose abundances in human motor neurons are sensitive to TDP-43 depletion. Notably, expression of STMN2, which encodes a microtubule regulator, declined after TDP-43 knockdown and TDP-43 mislocalization as well as in patient-specific motor neurons and postmortem patient spinal cord. STMN2 loss upon reduced TDP-43 function was due to altered splicing, which is functionally important, as we show STMN2 is necessary for normal axonal outgrowth and regeneration. Notably, post-translational stabilization of STMN2 rescued neurite outgrowth and axon regeneration deficits induced by TDP-43 depletion. We propose that restoring STMN2 expression warrants examination as a therapeutic strategy for ALS.Klim et al. illuminate pathomechanisms of ALS using pluripotent stem cells to identify transcripts altered in human motor neurons by perturbations to ALS protein TDP-43, finding the microtubule regulator STMN2 highly sensitive to TDP-43 malfunctions.

Prions are proteins that adopt alternative conformations that become self-propagating; the PrPScprion causes the rare human disorder Creutzfeldt–Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T–YFP) and TgM83+/−mice expressing α-synuclein (A53T). The TgM83+/−mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83+/+mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83+/−mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T–YFP in cultured cells, whereas none of six Parkinson’s disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83+/+mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron dysfunction disease that leads to paralysis and death. There is currently no established molecular pathogenesis pathway. Multiple proteins involved in RNA processing are linked to ALS, including FUS and TDP43, and we propose a disease mechanism in which loss of function of at least one of these proteins leads to an accumulation of transcription-associated DNA damage contributing to motor neuron cell death and progressive neurological symptoms. In support of this hypothesis, we find that FUS or TDP43 depletion leads to increased sensitivity to a transcription-arresting agent due to increased DNA damage. Thus, these proteins normally contribute to the prevention or repair of transcription-associated DNA damage. In addition, both FUS and TDP43 colocalize with active RNA polymerase II at sites of DNA damage along with the DNA damage repair protein, BRCA1, and FUS and TDP43 participate in the prevention or repair of R loop-associated DNA damage, a manifestation of aberrant transcription and/or RNA processing. Gaining a better understanding of the role(s) that FUS and TDP43 play in transcription-associated DNA damage could shed light on the mechanisms underlying ALS pathogenesis.

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimer’s disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to tau’s accumulation or what mechanisms might be involved. To explore these questions, we focus on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to weaken its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 reveals the mechanism of this clash, allowing design of a mutation (CHIP D134A ) that partially restores binding and turnover of pS416 tauC3. We confirm that, in our models, pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a molecular mechanism for how phosphorylation at a discrete site contributes to accumulation of a tau proteoform. Hyper-phosphorylated tau accumulates in Alzheimer’s disease; but the roles of these phosphorylation sites are unclear. Here the authors show that phosphorylation of a single site, pSer416, limits its turnover and promotes accumulation.

Increasingly, evidence argues that many neurodegenerative diseases, including progressive supranuclear palsy (PSP), are caused by prions, which are alternatively folded proteins undergoing selfpropagation. In earlier studies, PSP prions were detected by infecting human embryonic kidney (HEK) cells expressing a tau fragment [TauRD(LM)] fused to yellow fluorescent protein (YFP). Here, we report on an improved bioassay using selective precipitation of tau prions from human PSP brain homogenates before infection of the HEK cells. Tau prions were measured by counting the number of cells with TauRD(LM)–YFP aggregates using confocal fluorescence microscopy. In parallel studies, we fused α-synuclein to YFP to bioassay α-synuclein prions in the brains of patients who died of multiple system atrophy (MSA). Previously, MSA prion detection required ∼120 d for transmission into transgenic mice, whereas our cultured cell assay needed only 4 d. Variation in MSA prion levels in four different brain regions from three patients provided evidence for three different MSA prion strains. Attempts to demonstrate α-synuclein prions in brain homogenates from Parkinson’s disease patients were unsuccessful, identifying an important biological difference between the two synucleinopathies. Partial purification of tau and α-synuclein prions facilitated measuring the levels of these protein pathogens in human brains. Our studies should facilitate investigations of the pathogenesis of both tau and α-synuclein prion disorders as well as help decipher the basic biology of those prions that attack the CNS.

Multiple system atrophy (MSA), a progressive neurodegenerative disease characterized by autonomic dysfunction and motor impairment, is caused by the self-templated misfolding of the protein α-synuclein. With no treatment currently available, we sought to characterize the spread of α-synuclein in a transgenic mouse model of MSA prion propagation to support drug discovery programs for synucleinopathies. Brain homogenates from MSA patient samples or mouse-passaged MSA were inoculated either by standard freehand injection or stereotactically into TgM83+/- mice, which express human α-synuclein with the A53T mutation. Following disease onset, brains from the mice were tested for biologically active α-synuclein prions using a cell-based assay and examined for α-synuclein neuropathology. Inoculation studies using homogenates prepared from brain regions lacking detectable α-synuclein neuropathology transmitted neurological disease to mice. Terminal animals contained similar concentrations of α-synuclein prions; however, a time-course study where mice were terminated every five days through disease progression revealed that the kinetics of α-synuclein prion replication in the mice were variable. Stereotactic inoculation into the thalamus reduced variability in disease onset in the mice, although incubation times were consistent with standard inoculations. Using human samples with and without neuropathological lesions, we observed that α-synuclein prion formation precedes neuropathology in the brain, suggesting that disease in patients is not limited to brain regions containing neuropathological lesions.

Previously, we reported that intracranial inoculation of brain homogenate from multiple system atrophy (MSA) patient samples produces neurological disease in the transgenic (Tg) mouse model TgM83+/−, which uses the prion protein promoter to express human α-synuclein harboring the A53T mutation found in familial Parkinson’s disease (PD). In our studies, we inoculated MSA and control patient samples into Tg mice constructed using a P1 artificial chromosome to express wild-type (WT), A30P, and A53T human α-synuclein on a mouse α-synuclein knockout background [Tg(SNCA+/+)Nbm, Tg(SNCA*A30P+/+)Nbm, and Tg(SNCA*A53T+/+)Nbm]. In contrast to studies using TgM83+/− mice, motor deficits were not observed by 330–400 days in any of the Tg(SNCA)Nbm mice after inoculation with MSA brain homogenates. However, using a cell-based bioassay to measure α-synuclein prions, we found brain homogenates from Tg(SNCA*A53T+/+)Nbm mice inoculated with MSA patient samples contained α-synuclein prions, whereas control mice did not. Moreover, these α-synuclein aggregates retained the biological and biochemical characteristics of the α-synuclein prions in MSA patient samples. Intriguingly, Tg(SNCA*A53T+/+)Nbm mice developed α-synuclein pathology in neurons and astrocytes throughout the limbic system. This finding is in contrast to MSA-inoculated TgM83+/− mice, which develop exclusively neuronal α-synuclein aggregates in the hindbrain that cause motor deficits with advanced disease. In a crossover experiment, we inoculated TgM83+/− mice with brain homogenate from two MSA patient samples or one control sample first inoculated, or passaged, in Tg(SNCA*A53T+/+)Nbm animals. Additionally, we performed the reverse experiment by inoculating Tg(SNCA*A53T+/+)Nbm mice with brain homogenate from the same two MSA samples and one control sample first passaged in TgM83+/− animals. The TgM83+/− mice inoculated with mouse-passaged MSA developed motor dysfunction and α-synuclein prions, whereas the mouse-passaged control sample had no effect. Similarly, the mouse-passaged MSA samples induced α-synuclein prion formation in Tg(SNCA*A53T+/+)Nbm mice, but the mouse-passaged control sample did not. The confirmed transmission of α-synuclein prions to a second synucleinopathy model and the ability to propagate prions between two distinct mouse lines while retaining strain-specific properties provides compelling evidence that MSA is a prion disease.

In the neurodegenerative disease multiple system atrophy (MSA), α-synuclein misfolds into a self-templating conformation to become a prion. To compare the biological activity of α-synuclein prions in MSA and Parkinson’s disease (PD), we developed nine α-synuclein−YFP cell lines expressing point mutations responsible for inherited PD. MSA prions robustly infected wild-type, A30P, and A53T α-synuclein–YFP cells, but they were unable to replicate in cells expressing the E46K mutation. Coexpression of the A53T and E46K mutations was unable to rescue MSA prion infection in vitro, establishing that MSA α-synuclein prions are conformationally distinct from the misfolded α-synuclein in PD patients. This observation may have profound implications for developing treatments for neurodegenerative diseases.

The study of amyotrophic lateral sclerosis (ALS) and potential interventions would be facilitated if motor axon degeneration could be more readily visualized. Here we demonstrate that stimulated Raman scattering (SRS) microscopy could be used to sensitively monitor peripheral nerve degeneration in ALS mouse models and ALS autopsy materials. Three-dimensional imaging of pre-symptomatic SOD1 mouse models and data processing by a correlation-based algorithm revealed that significant degeneration of peripheral nerves could be detected coincidentally with the earliest detectable signs of muscle denervation and preceded physiologically measurable motor function decline. We also found that peripheral degeneration was an early event in FUS as well as C9ORF72 repeat expansion models of ALS, and that serial imaging allowed long-term observation of disease progression and drug effects in living animals. Our study demonstrates that SRS imaging is a sensitive and quantitative means of measuring disease progression, greatly facilitating future studies of disease mechanisms and candidate therapeutics. Sensitive and label-free imaging methods to visualize nerve degeneration are currently lacking. Here authors show that stimulated Raman scattering (SRS) microscopy can be used to monitor peripheral nerve degeneration in mouse models of amyotrophic lateral sclerosis (ALS) and in postmortem tissue from ALS patients.

In multiple system atrophy (MSA), the α-synuclein protein misfolds into a self-templating prion conformation that spreads throughout the brain, leading to progressive neurodegeneration. While the E46K mutation in α-synuclein causes familial Parkinson’s disease (PD), we previously discovered that this mutation blocks in vitro propagation of MSA prions. Recent studies by others indicate that α-synuclein adopts a misfolded conformation in MSA in which a Greek key motif is stabilized by an intramolecular salt bridge between residues E46 and K80. Hypothesizing that the E46K mutation impedes salt bridge formation and, therefore, exerts a selective pressure that can modulate α-synuclein strain propagation, we asked whether three distinct α-synuclein prion strains could propagate in TgM47 +/- mice, which express human α-synuclein with the E46K mutation. Following intracranial injection of these strains, TgM47 +/- mice were resistant to MSA prion transmission, whereas recombinant E46K preformed fibrils (PFFs) transmitted neurological disease to mice and induced the formation of phosphorylated α-synuclein neuropathology. In contrast, heterotypic seeding following wild-type (WT) PFF–inoculation resulted in preclinical α-synuclein prion propagation. Moreover, when we inoculated TgM20 +/- mice, which express WT human α-synuclein, with E46K PFFs, we observed delayed transmission kinetics with an incomplete attack rate. These findings suggest that the E46K mutation constrains the number of α-synuclein prion conformations that can propagate in TgM47 +/- mice, expanding our understanding of the selective pressures that impact α-synuclein prion replication.