The selective loss of substantia nigra (SN) dopaminergic (DA) neurons in neurodegenerative disorders including Parkinson’s disease, multiple system atrophy, progressive supranuclear palsy, and corticobasal degeneration is well known. These diseases are characterized by the abnormal aggregation of alpha-synuclein and tau protein in the form of cytoplasmic and neuritic inclusion bodies. SN DA neurons also degenerate in diseases characterized by intranuclear inclusion bodies comprised of polyglutamine expanded proteins, such as the spinocerebellar ataxias (Woulfe, 2007). These inclusions stain for the proteolysis-associated proteins ubiquitin and p62, which serve as markers for pathological protein aggregates. There is unequivocal evidence that SN DA neurons also undergo prominent loss of function during aging in non-diseased individuals (Collier et al., 2011) although there is controversy concerning whether there is overt neuronal loss (see (Fearnley and Lees, 1991, Ma et al., 1999) (McGeer et al., 1977) but also (Alladi et al., 2009; Chu et al., 2002; Kubis et al., 2000)). These degenerative changes not only affect motor performance in older adults, but lower the threshold for the clinical expression of Parkinson’s disease (Collier et al., 2011). By analogy with the neurodegenerative diseases listed above, the aging SN in these non-diseased individuals is characterized by the accumulation of p62- and ubiquitin-immunoreactive inclusions bodies, such as intranuclear Marinesco bodies (MBs) (Dickson et al., 1990, Gray et al., 2003) and neuritic inclusions (Braak et al., 2013). Importantly however, these do not stain for alpha-synuclein, tau, or known polyglutamine proteins (Dickson et al., 1990, Gray et al., 2003). This implies distinct pathogenetic mechanisms underlying SN DA neuron dysfunction in aging versus neurodegenerative disease. Moreover, the existence of these structures suggests a role for the aggregation of hitherto unidentified protein(s) that are not linked with canonical neurodegenerative diseases in SN DA neuron degeneration that is strictly age-related.

Inosine monophosphate dehydrogenase (IMPDH), is the rate-limiting enzyme in the de novo synthesis of the guanine-based purine nucleotides GMP, GDP, and GTP (Fig. 1). IMPDH protein is encoded by two genes, IMPDH1 and IMPDH2 (Natsumeda et al., 1990). IMPDH1 is expressed in retina (Senda and Natsumeda, 1994) and its mutations cause retinitis pigmentosa (McGrew and Hedstrom, 2012) while IMPDH2 is expressed ubiquitously in all tissues, including the brain (Senda and Natsumeda, 1994), and its mutations have been linked with neurodevelopmental disorders (Kuukasjarvi et al., 2021, O'Neill et al., 2023, Zech et al., 2020). Purines are ubiquitous biomolecules that are essential for life. They are incorporated into RNA and DNA, provide the energy currency of cells, are used as signalling molecules, and are incorporated into co-enzymes. In response to purine limitation, mTOR signalling, and mitochondrial dysfunction, enzymes involved in the de novo purine pathway are assembled by liquid-liquid phase separation into microscopically visible, supramolecular membrane less organelles of at least nine enzymes called "purinosomes" (An et al., 2008, French et al., 2016). The nine enzymes partitioned to this "metabolon" structure, by virtue of their physical proximity, are capable of efficiently catalyzing the generation of AMP and GMP in 14 sequential substrate channeling steps, thereby achieving greater pathway flux in response to metabolic demand (Pareek et al., 2020).

In addition to purinosomes, a subset of these enzymes, including IMPDH, are subject to an additional level of supramolecular regulation; the formation of mesoscale linear filamentous assemblies as a biological regulatory mechanism to co-ordinate enzyme activity with cellular requirements (Aughey and Liu, 2015, Johnson and Kollman, 2020, Lynch et al., 2020, Simonet et al., 2020). The relationship between filament formation and enzyme activity is complex as both active and inactive octomeric conformers are present within individual filaments (Johnson and Kollman, 2020). Current consensus supports a model whereby filament formation correlates with increased enzyme activity by resisting feedback inhibition by GTP (Johnson and Kollman, 2020). This adds nuance to purinosome function; permitting ongoing GTP synthesis, even when it is relatively plentiful, under conditions of increased GTP demand. Individual IMPDH filaments can aggregate by lateral alignment to form larger, microscopically-visible filament bundles which have been variously referred to as ”IMPDH filaments” "rods and ring structures" or "cytoophidia" (Greek for "cellular snakes"; reviewed in (Calise and Chan, 2020; Liu, 2016)). The formation of these structures at steady state and in response to metabolic perturbations has been described in a variety of cell types both in vitro and in vivo (Calise and Chan, 2020). Although filamentation subserves an adaptive function to support GTP synthesis, aberrant oligomerization of IMPDH may have detrimental consequences. Indeed, aberrant IMPDH1 aggregation with the formation of long-lived, toxic cytoophidia has been implicated as a pathogenetic mechanism in IMPDH1-linked retinitis pigmentosa (Aherne et al., 2004, Keppeke et al., 2023). Moreover, under conditions of over-expression, the phase separation process of IMPDH can extend beyond the formation of linear filaments to form large, potentially cytotoxic rigid intracellular crystals (Nass et al., 2020).

The aim of the present study is to examine a previously unknown morphological feature of human SN DA neurons; namely the presence of cytoplasmic IMPDH filaments and intranuclear IMPDH inclusion bodies. Specifically, we sought to interrogate their occurrence during normal development and aging under conditions lacking tau- or alpha-synuclein pathology. Our results provide evidence for a novel intranuclear protein aggregation mechanism contributing to aging-related changes in the SN that is distinct from those associated with known neurodegeneration-associated proteins.