*3.5. Poly(ADP-Ribosylation)*

Poly(ADP-ribosyl)ation or PARylation is a reversible covalent addition of multiple NAD-derived ADP-ribose (ADPr) molecules to a protein [78]. ADPr units can be added to glutamate, aspartate, lysine, arginine, or serine residues by poly(ADP-ribose) polymerases (PARPs) and removed by PAR glycohydrolases (PARGs) [78]. Aside from the physical addition of ADPr units, polyADP-ribose (PAR) molecules are freely synthesized polymers that can modify phase separation of some IDPs [79]. PAR is a multivalent, anionic, nucleic acid-mimicking (similar to RNA) biopolymer that can be bound by phase separating proteins [2]. Cellular stress conditions and DNA damage have been shown to cause an upregulation of PAR synthesis [79]. PAR, PARPs, and PARGs have all been shown to play a regulatory role in SG dynamics [80].

The SG component hnRNPA1 contains both a PAR-binding domain and a PARylation site at Lysine 298 within a glycine rich IDR. PARylation at hnRNPA1 Lysine 298 is important for hnRNPA1 nucleocytoplasmic shuttling, a necessary step for localization to SGs following cellular stress [81]. Interestingly, like numerous other proteins, hnRNPA1 contains a PAR-binding motif (PBM). In vitro experiments showed hnRNPA1 phase separation increasing in response to increased PAR concentration in solution. Mutating the hnRNPA1 PBM resulted in no phase separation in the presence of PAR, implying this interaction is domain specific. TDP-43 also contains a PBM and co-phase separates with hnRNPA1 in vitro and in SGs [81,82]. In vitro, PAR binding via hnRNPA1 PBM is necessary for the co-phase separation of TDP-43 and hnRNPA1 in low-salt concentrations. In cells, both TDP-43 and hnRNPA1 need functional PBMs to localize to SGs, highlighting the role of PAR in protein–protein interaction and phase transition [81,82].

#### **4. Membraneless Organelles and Neurodegenerative Diseases**

A connection between MLOs and neurodegenerative disease has been widely observed [83]. Specifically, many proteins that are genetically or histopathologically linked to neurodegeneration are also found in neuronal MLOs [18]. Likewise, proteins with intrinsically disordered PrLDs are notoriously disproportionately linked to neurodegenerative disease [84], and many of these proteins are both capable of undergoing LLPS and frequently found within inclusions of diseased neurons [85].

Why do the same proteins that functionally undergo LLPS appear to adopt pathological meso-scale aggregates in cells? A leading hypothesis is proteins within MLOs may undergo additional transitions into oligomeric species, solid-phase aggregates [86], or droplet-like structures with dramatically different material properties [17] (Figure 1). For example, expression of an ALS-linked TDP-43 mutant results in an MLO that is more viscous and resistant to solvation, suggesting it has a stabilized internal structure [19]. Similarly, in vitro, ALS-mutant FUS can transition from a droplet state into a solid aggregate more rapidly than wild-type FUS [42]. Once formed, such aggregates are thought to be detrimental to cell function and contribute to neuronal degeneration. Possibly, the high concentration of specific proteins within MLOs may potentiate these stochastic, irreversible phase transitions. Since disease-linked proteins like tau and TDP-43 are hyper- and multi-phosphorylated, respectively, within neuronal cytoplasmic inclusions, it is possible the PTMs are facilitating solid-phase transitions; alternatively, the PTMs may simply mark failed attempts at solubilization.

In the case of many IDRs, the abundance of hydrophilic amino acids and lack of stable tertiary structures may facilitate solid-phase transitions into highly ordered amyloid conformations. Amyloid is a well-ordered, filamentous polymeric state composed of a single protein species, much like a one-dimensional crystal [87]. It usually consists of polypeptides aligning in parallel in-register beta sheets [88] and is notoriously difficult to solubilize. MLOs may provide an environment in which some enriched IDR-containing proteins can stochastically adopt amyloid-like conformations, thus explaining the presence of certain MLO-linked proteins in pathological neuronal inclusions. Examples include tau

(Alzheimer's disease), TDP-43 (ALS), and FUS (frontotemporal dementia). Importantly, crystal-like arrangements would be disrupted by PTMs occurring within the structural core of amyloid [89]; thus, targeting specific modifying enzymes could offer a viable therapeutic strategy for neurodegenerative disorders that feature solid-phase inclusions.
