Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder categorized to the progressive motor neuron disease, and its incident is most frequent in sexagenarians and septuagenarians (1, 2). An epidemiological study showed that 1.68 per 100,000 person-years suffered from this disease in worldwide with substantial variations in region (3). Sporadic ALS where the ALS onset is independently of hereditary traits accounts for 70–80% of all of the ALS cases, confounding the genetic prediction of future ALS onset (4). Due to the absence of effective biomarkers for ALS, its diagnosis hinges on the indirect approaches by checking clinical symptoms and measuring muscle action potential amplitudes, which results in the delayed therapeutic intervention with limited number of medical options (5). To tackle these issues, a number of ALS-causative proteins including C9orf72, TDP-43 and FUS have been identified and studied for the development of ALS therapeutic agents selectively targeting these molecules (6, 7). In addition, the discovery of potentially diagnostic biomarkers for ALS such as Neurofilament L (NfL) facilitates the biofluid-based noninvasive approaches for ALS diagnosis (8–10). Yet, there is still a lack of molecular information with regard to the ALS pathology, and thus its features in molecular machinery should be elucidated more clearly for liberating humanity from the agony of this disease.

Inositol pyrophosphate exists in a wide variety of organisms from slime molds and fungi to mammals and is involved in numerous cellular processes including intracellular signaling (11–13). Diphosphoinositol pentakisphosphate (a.k.a. IP₇), a representative inositol pyrophosphate in mammals, is synthesized from the precursor molecule inositol hexakisphosphate (a.k.a. IP₆) by inositol hexakisphosphate kinases (IP6Ks). So far, several lines of evidence suggested pathological roles of IP6Ks and IP₇ in stress response and neurodegeneration. IP6K2, one of the major IP₇-synthesizing kinase in mammals, was characterized as a cell death mediator (14) and IP₇ facilitates cellular apoptosis by reactive oxygen treatment (15). Considering our previous observations of IP6K2 mRNA induction during presymptomatic disease state of ALS (16) and IP6K2 modulatory role in TDP-43-mediated cellular apoptosis (17), these facts collectively imply the notion that IP₇ would be induced in aberrant motor neuron of ALS disease state. However, none of the direct evidences has not been obtained due to lack of technologies directly detecting and quantifying IP₇. Recently, we developed an analytical protocol directly and selectively detecting IP₇ in mammalian tissues (18, 19), unlocking the direct evaluation of this molecule in various clinical biopsies.

In this study, we analyzed endogenous IP₇ and its precursor IP₆ in ALS model mice as well as human ALS patients by an originally-designed liquid chromatography mass spectrometry (LC-MS) protocol and assessed if ALS disease state would be accompanied by altered IP₇ level.

While a number of studies have identified causative proteins and potential biomarkers for ALS so far (6), such exploring efforts still continue to understand the molecular machinery of this disease more precisely. Several studies reported the implicit findings that inositol pyrophosphate IP₇ might be associated with ALS pathogenesis (14–17), but there has been no direct evidence proving this notion. In this study, we analyzed IP₇ and its precursor IP₆ in ALS model mice and ALS patients by an originally-designed LC-MS protocol (18, 19) to directly examine the relationship between IP₇ and ALS.

We used a canonical ALS mouse model SOD1(G93A) TG mice to analyze IP₇ level and metabolism before and after ALS onset and found that IP₇ level significantly increased in the spinal cord of the TG mice at the late stage of the disease (Figures 1B, C). The spinal cord of the TG mice showed slightly but not significantly elevated IP₇ metabolism (IP₇/IP₆ ratio) due to the up-regulation of IP₆ level concomitantly with IP₇ elevation, implying dysregulation of the metabolic pathway in lower inositol phosphates (IPs). A recent transcriptome analysis using the TG mice exhibited that mRNA levels of certain genes involved in phosphatidyl inositol metabolic process such as INPP5D (inositol polyphosphate-5-phosphatase D) and INPPL1 (inositol polyphosphate phosphatase like 1) was changed in the spinal cord at ALS disease state (26). Such alteration in inositol phospholipid pathway might lead to the dysregulated metabolism of IP₇ and other lower inositol phosphates. Similar with the results of this rodent ALS model, significant elevation of IP₇ level and metabolism was observed in the postmortem lumber of human ALS patients (Figures 2B, C). Considering our previous data that the transcript level of IP₇-synthesizing enzyme IP6K2 increased in the spinal cord of ALS disease state (16), transcriptional activation of this enzyme is likely to contributes to IP₇ elevation in the spinal cord of ALS.

The molecular mechanism underlying IP₇ induction in the ALS condition is still elusive, but IP₇ and its synthesizing kinase IP6Ks has been shown to associate with several neurodegeneration-related proteins. In our previous report, IP6K2 interacts with TDP-43 and promotes TDP-43-inducing cell death (17). A recent study identified IP₇ kinase PPIP5K as an α-synuclein neurotoxicity modulator by functional RNAi screening using nematode Perkinson's disease model (27). Moreover, IP6K and IP₇ facilitate the formation of aberrant protein-RNA aggregates inducing neurotoxicity in various neurodegenerative disorders (28) at least via promoting the interaction of RNA-binding proteins (for IP6K) and inhibiting the 5′-decapping reaction of non-translated mRNAs (for IP₇) (29, 30). In addition, IP₇ competitively binds to AKT and inhibits its downstream signaling including mTOR, a key regulator of cell survival (31, 32) (Information of proteins associating with IP₆ was summarized in Supplementary Figure 4). These pieces of knowledge suggest that IP₇ could regulate various neurodegenerative-related proteins in a multifaceted manner. However, we did not investigate pathobiological role of IP₇ in the CNS of ALS model mice because the major barrier for studying IP₇ functions in mouse models is the difficulty to efficiently inhibit IP₇ production in vivo. Two genes IP6K1 and IP6K2 are responsible for IP₇ production, but the deletion of both genes results in embryonic death and the deletion of single gene partially inhibits its production in the CNS as shown in our recent report (19). Recent report showed that a novel IP6K inhibitor efficiently blocks IP₇ production in vivo (24), which will enable to investigate the role of IP₇ in ALS using disease model mice. Further investigation will be warranted to prove the notion that elevated IP₇ is associated with progressive degeneration of spinal motor neurons during ALS.

Since several ALS-associated molecules such as NfL and TDP-43 are promising for applying biofluid-based ALS diagnostic markers (23), we examined the applicability of IP₇ in such diagnostic approach using peripheral blood and CSF. Among human peripheral blood fractions, PBMCs possessed most abundant IP₆ and IP₇ (Supplementary Figure 1), but the levels of these molecules in PBMCs could not differentiate ALS patients from age-matched healthy individuals (Figure 3C), suggesting the unfeasibility of peripheral blood IP₇ for usage as an ALS diagnostic marker. In addition, our LC-MS analysis could not detect IP₇ and IP₆ in human CSF, suggesting that the abundances of these molecules in CSF are less than 0.3 μM considering the lower limit of detection in our LC-MS protocol (18, 19). It would be necessary to evaluate CSF IP₇ level by more sensitive protocol such as capillary electrophoresis-mass spectrometry (CE-MS) (33) or develop a technology whereby spinal cord IP₇ is measured non-invasively for considering the application of IP₇ for ALS diagnosis.

Although this study focused at the relationship between IP₇ and ALS in this study, it is meaningful to elucidate if IP₇ level and its metabolism would be altered in other neurodegenerative disorders. So far, IP₇ has been implied to be associated with certain neurodegenerative diseases such as Huntington's disease (34) and Alzheimer's disease (35). Sensitive mass spectrometric analysis of IP₇ using clinical biopsies as well as mouse disease models would unveil a pathobiological role of IP₇ in such diseases in future.

Recently, CE-MS analysis clarified that several IP₇ isotypes such as 4/6-IP₇ and 1/3-IP₇ constitute mammalian IP₇ in addition to 5-IP₇, an already-known isotype of mammalian IP₇ (25). Due to the technical limitation of LC-MS, we could not determine if the IP₇ status would be affected during ALS at the level of isotype. Further investigation is required which isotypes of IP₇ is elevated in the spinal cord of ALS disease state.

We demonstrate for the first time that IP₇ level and/or its metabolism (IP₇/IP₆ ratio) were significantly elevated in mouse and/or human ALS spinal cord compared with neurologically normal counterparts in a direct way. On the other hand, this study showed several limitations. First, the sample number of postmortem ALS spinal cord isn't necessarily sufficient for the statistical analysis. Secondly, we failed to demonstrate IP₇ level and its metabolism as biofluid-based diagnostic parameters for ALS. Lastly, we could not clarify the molecular basis and pathophysiological significance of IP₇ elevation during ALS disease state. We believe that further investigation of IP₇ function in ALS pathology will lead to the development of novel diagnostic and therapeutic approach for this incurable disease.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author/s.

The studies involving humans were approved by the Clinical Investigation Committee at Tokai University School of Medicine. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. The animal study was approved by the Institutional Animal Care and Use Committee at Tokai University. The study was conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

MI: Formal analysis, Investigation, Methodology, Visualization, Writing—original draft, Writing—review & editing. NF: Investigation, Writing—review & editing. SK: Investigation, Writing—review & editing. MTan: Formal analysis, Writing—review & editing. MTak: Funding acquisition, Resources, Writing—review & editing. BM: Resources, Writing—review & editing. YS: Funding acquisition, Resources, Writing—review & editing. AM: Writing—review & editing. TN: Writing—review & editing. SN: Writing—review & editing. NS: Writing—review & editing. AK: Funding acquisition, Resources, Writing—review & editing. EN: Conceptualization, Funding acquisition, Project administration, Supervision, Writing—review & editing.