Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear TDP-43. Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-mRNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from sporadic ALS patients and familial ALS patients with expansion in C9orf72, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS/FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability.

The RNA binding protein TDP-43 forms intranuclear or cytoplasmic aggregates in age-related neurodegenerative diseases. In this study, we found that RNA binding–deficient TDP-43 (produced by neurodegeneration-causing mutations or posttranslational acetylation in its RNA recognition motifs) drove TDP-43 demixing into intranuclear liquid spherical shells with liquid cores. These droplets, which we named “anisosomes”, have shells that exhibit birefringence, thus indicating liquid crystal formation. Guided by mathematical modeling, we identified the primary components of the liquid core to be HSP70 family chaperones, whose adenosine triphosphate (ATP)–dependent activity maintained the liquidity of shells and cores. In vivo proteasome inhibition within neurons, to mimic aging-related reduction of proteasome activity, induced TDP-43–containing anisosomes. These structures converted to aggregates when ATP levels were reduced. Thus, acetylation, HSP70, and proteasome activities regulate TDP-43 phase separation and conversion into a gel or solid phase.

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One Sentence Summary:Acetylation of TDP-43 drives its phase separation into spherical annuli that form a liquid-insidea-liquid-inside-a-liquid. AbstractThe RNA binding protein TDP-43 naturally phase separates within cell nuclei and forms cytoplasmic aggregates in age-related neurodegenerative diseases. Here we show that acetylation-mediated inhibition of TDP-43 binding to RNA produces co-de-mixing of acetylated and unmodified TDP-43 into symmetrical, intranuclear spherical annuli whose shells and cores have liquid properties. Shells are anisotropic, like liquid crystals. Consistent with our modelling predictions that annulus formation is driven by components with strong self-interactions but weak interaction with TDP-43, the major components of annuli cores are identified to be HSP70 family proteins, whose chaperone activity is required to maintain liquidity of the core. Proteasome inhibition, mimicking reduction in proteasome activity during aging, induces TDP-43-containing annuli in neurons in rodents. Thus, we identify that TDP-43 phase separation is regulated by acetylation, proteolysis, and ATPase-dependent chaperone activity of HSP70.A seminal discovery in the last decade has been recognition that large biological molecules, especially RNA binding proteins, can undergo liquid-liquid phase separation (LLPS), resembling oil droplets in vinegar. Under certain physical conditions, proteins, nucleic acids, or a mixture of both in a complex solution can form two phases, a condensed de-mixed phase and more dilute aqueous phase (1). Inside the cell, proteins and/or nucleic acids de-mix into a condensed phase to form membraneless organelles which have been proposed to promote biological functions (2).One membraneless organelle is the nucleolus, first described in the early 1800's and now recognized to be composed of proteins that undergo LLPS (3-5). Discovery that P-granules are de-mixed compartments with liquid behaviour brought widespread recognition to LLPS and its ability to mediate subcellular compartmentalization in a biological context (6). Phase separation has been also proposed for heterochromatin and RNA bodies (1, 6, 7). LLPS potentially underlies the operational principle governing formation of important organelles and structures, such as centrosomes, nuclear pore complexes, and super enhancers (8-10).Few mechanisms to modulate intracellular LLPS have been identified. Disease-causing mutations of proteins such as FUS (11) and HNRNPA2B1 (12) alter their LLPS behaviors in vitro, but the physiological or pathological relevance of their intracellular LLPS has not been fully elucidated. Random, multivalent interactions among intrinsically disordered, low complexity domains (LCDs) of proteins are a major driving force for LLPS in vitro. Oligomerization of other domains is thought to be required for LLPS in vivo (13). The inherent randomness of multivalent interactions (14) favors a model of disordered alignment -a conventional liquid phase in which molecules randomly move. The evidence is compelling that multi...