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The progressive degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is accompanied by the formation of a broad array of cytoplasmic and nuclear neuronal inclusions (protein aggregates) largely containing RNA-binding proteins such as TAR DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS). This process is driven by a liquid-to-solid phase separation generally from proteins in membrane-less organelles giving rise to pathological biomolecular condensates. The formation of these protein aggregates suggests a fundamental alteration in the mRNA expression or the levels of the proteins involved. Considering the role of the epigenome in gene expression, alterations in DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs, and RNA modifications become highly relevant to understanding how this pathological process takes effect. In this review, we explore the evidence that links epigenetic mechanisms with the formation of protein aggregates in ALS. We propose that a greater understanding of the role of the epigenome and how this inter-relates with the formation of pathological LLPS in ALS will provide an attractive therapeutic target.

Abstract Ethical animal use follows the 3R’s: Replacement, Reduction and Refinement. Here, we present the use of simultaneous jugular vein and cisterna magna catheterization via a port system in rats for repeated fluid sampling for 14 consecutive days without loss of catheter patency. This technique allows repeated intra-animal sampling without anesthesia and, if used with pooling samples from a cohort of animals, replaces the need for terminal collections for sufficient sample volumes.

Abstract Background As cancer therapies have improved, spinal metastases are increasingly common. Resulting complications have a significant impact on patient's quality of life. Optimal methods of surveillance and avoidance of neurologic deficits are understudied. This study compares the clinical course of patients who initially presented to the emergency department (ED) versus a multidisciplinary spine oncology clinic and who underwent stereotactic body radiation therapy (SBRT) secondary to progression/presentation of metastatic spine disease. Methods We performed a retrospective analysis of a prospectively maintained database of adult oncologic patients who underwent spinal SBRT at a single hospital from 2010 to 2021. Descriptive statistics and survival analyses were performed. Results We identified 498 spinal radiographic treatment sites in 390 patients. Of these patients, 118 (30.3%) presented to the ED. Patients presenting to the ED compared to the clinic had significantly more severe spinal compression (52.5% vs. 11.7%; p

Paraspeckles are nuclear condensates formed by NEAT1_2 lncRNA and different RNA-binding proteins. In general, these membraneless organelles function in the regulation of gene expression and translation and in miRNA processing, and in doing this, they regulate cellular homeostasis and mediate pro-survival in the cell. Despite evidence showing the importance of paraspeckles in the stress response, the dynamics of paraspeckles and their components under conditions of osmotic stress remain unknown. We exposed HEK293T cells to sorbitol and examined NEAT1_2 expression using real-time PCR. Localization and quantification of the main paraspeckle components, NEAT1_2, PSPC1, NONO, and SFPQ, in different cellular compartments was performed using smFISH and immunofluorescence. Our findings showed a significant decrease in total NEAT1_2 expression in cells after osmotic stress. Sorbitol shifted the subcellular localization of NEAT1_2, PSPC1, NONO, and SFPQ from the nucleus to the cytoplasm and decreased the number and size of NEAT1_2 foci in the nucleus. PSPC1 formed immunoreactive cytoplasmic fibrils under conditions of osmotic stress, which slowly disassembled under recovery. Our study deepens the paraspeckle dynamics in response to stress, suggesting a novel role for NEAT1_2 in the cytoplasm in osmotic stress and physiological conditions.

Phosphorylated microtubule-associated protein tau (tau) aggregates are a pathological hallmark of various neurodegenerative diseases, including chronic traumatic encephalopathy and amyotrophic lateral sclerosis with cognitive impairment. While there are many residues phosphorylated on tau, phosphorylation of threonine 175 (pThr175 tau) has been shown to initiate fibril formation in vitro and is present in pathological tau aggregates in vivo. Given this, preventing Thr175 tau phosphorylation presents a potential approach to reduce fibril formation; however, the kinase(s) acting on Thr175 are not yet fully defined. Using a single controlled cortical impact rodent model of traumatic brain injury (TBI), which rapidly induces Thr175 tau phosphorylation, we observed an upregulation and alteration in subcellular localization of leucine-rich repeat kinase 2 (LRRK2), a kinase that has been implicated in tau phosphorylation. LRRK2 upregulation was evident by one-day post-injury and persisted to day 10. The most notable changes were observed in microglia at the site of injury in the cortex. To determine if the appearance of pThr175 tau was causally related to the upregulation of LRRK2 expression, we examined the ability of LRRK2 to phosphorylate Thr175in vitro by co-transfecting 2N4R human WT-tau with either LRRK2-WT, constitutively-active LRRK2-G2019S or inactive LRRK2-3XKD. We found no significant difference in the level of pThr175 tau between the overexpression of LRRK2-WT, -G2019S or -3XKD, suggesting LRRK2 does not phosphorylate tau at Thr175. Further, downstream events known to follow Thr175 phosphorylation and known to be associated with pathological tau fibril formation (pSer9-GSK3β and pThr231 tau induction) also remained unchanged. We conclude that while LRRK2 expression is altered in TBI, it does not contribute directly to pThr175 tau generation.

Abstract We have previously reported long-term changes in the brains of non-concussed varsity rugby players using magnetic resonance spectroscopy (MRS), diffusion tensor imaging (DTI) and functional magnetic imaging (fMRI). Others have reported cognitive deficits in contact sport athletes that have not met the diagnostic criteria for concussion. These results suggest that repetitive mild traumatic brain injuries (rmTBIs) that are not severe enough to meet the diagnostic threshold for concussion, produce long-term consequences. We sought to characterize the neuroimaging, cognitive, pathological and metabolomic changes in a mouse model of rmTBI. Using a closed-skull model of mTBI that when scaled to human leads to rotational and linear accelerations far below what has been reported for sports concussion athletes, we found that 5 daily mTBIs triggered two temporally distinct types of pathological changes. First, during the first days and weeks after injury, the rmTBI produced diffuse axonal injury, a transient inflammatory response and changes in diffusion tensor imaging (DTI) that resolved with time. Second, the rmTBI led to pathological changes that were evident months after the injury including: changes in magnetic resonance spectroscopy (MRS), altered levels of synaptic proteins, behavioural deficits in attention and spatial memory, accumulations of pathologically phosphorylated tau, altered blood metabolomic profiles and white matter ultrastructural abnormalities. These results indicate that exceedingly mild rmTBI, in mice, triggers processes with pathological consequences observable months after the initial injury.

Abstract Background Numerous publications suggest an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports have employed immunohistochemistry, in situ hybridization and/or PCR, which are susceptible to specificity artifacts. Methods We investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide a high degree of both sensitivity and specificity. We quantified viral RNA expression for 740 viruses in 28 IPF patient lung biopsy samples and 20 controls. Key RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, HCV, herpesvirus saimiri and HERV-K). Results We identified sporadic low-level evidence of viral infections in our lung tissue specimens, but did not find a statistical difference for expression of any virus, including EBV, herpesvirus saimiri and HERV-K, between IPF and control lungs. Conclusions To the best of our knowledge, this is the first publication that employs RNA-seq to assess whether viral infections are linked to the pathogenesis of IPF. Our results do not address the role of viral infection in acute exacerbations of IPF, however, this analysis patently did not support an association between herpes virus detection and IPF.

Despite the wide application of the magnetic resonance imaging (MRI) technique, there are no widely used standards on naming and describing MRI sequences. The absence of consistent naming conventions presents a major challenge in automating image processing since most MRI software require a priori knowledge of the type of the MRI sequences to be processed. This issue becomes increasingly critical with the current efforts toward open-sharing of MRI data in the neuroscience community. This manuscript reports an MRI sequence detection method using imaging metadata and a supervised machine learning technique. Three datasets from the Brain Center for Ontario Data Exploration (Brain-CODE) data platform, each involving MRI data from multiple research institutes, are used to build and test our model. The preliminary results show that a random forest model can be trained to accurately identify MRI sequence types, and to recognize MRI scans that do not belong to any of the known sequence types. Therefore the proposed approach can be used to automate processing of MRI data that involves a large number of variations in sequence names, and to help standardize sequence naming in ongoing data collections. This study highlights the potential of the machine learning approaches in helping manage health data.

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease with no known etiology. The formation of pathological protein inclusions, including RNA-binding proteins such as TDP-43 and rho guanine nucleotide exchange factor (RGNEF) are a hallmark of ALS. Despite intensive research, the mechanisms behind protein aggregate formation in ALS remains unclear. We have investigated the role of metabolic stress in protein aggregate formation analyzing how it is relevant to the co-aggregation observed between RGNEF and TDP-43 in motor neurons of ALS patients. Metabolic stress was able to induce formation of micronuclei, small nuclear fragments, in cultured cells. Notably, we observed the formation TDP-43 protein inclusions within micronuclei that co-aggregate with RGNEF and can be released to the cytoplasm. We observed that the leucine-rich domain of RGNEF is critical for its interaction with TDP-43 and localization in micronuclei. Finally, we described that micronuclei-like structures can be found in brain and spinal cord of ALS patients. This work is the first description of protein inclusion formation within micronuclei which also is linked with a neurodegenerative disease. The formation of TDP-43 inclusions within micronuclei induced by metabolic stress is a novel mechanism of protein aggregate formation which may have broad relevance for ALS and other neurodegenerative diseases.

Abstract Although it has been suggested that the co-expression of multiple pathological proteins associated with neurodegeneration may act synergistically to induce more widespread neuropathology, experimental evidence of this is sparse. We have previously shown that the expression of Thr175Asp-tau (tauT175D) using somatic gene transfer with a stereotaxically-injected recombinant adeno-associated virus (rAAV9) vector induces tau pathology in rat hippocampus. In this study, we have examined whether the co-expression of human tauT175D with mutant human TDP-43 (TDP-43M337V) will act synergistically. Transgenic female Sprague-Dawley rats that inducibly express mutant human TDP-43M337V using the choline acetyltransferase (ChAT) tetracycline response element (TRE) driver with activity modulating tetracycline-controlled transactivator (tTA) were utilized in these studies. Adult rats were injected with GFP-tagged tau protein constructs in a rAAV9 vector through bilateral stereotaxic injection into the hippocampus. Injected tau constructs were: wild-type GFP-tagged 2N4R human tau (tauWT; n = 8), GFP-tagged tauT175D 2N4R human tau (tauT175D, pseudophosphorylated, toxic variant, n = 8), and GFP (control, n = 8). Six months post-injection, mutant TDP-43M337V expression was induced for 30 days. Behaviour testing identified motor deficits within 3 weeks after TDP-43 expression irrespective of tau expression, though social behaviour and sensorimotor gating remained unchanged. Increased tau pathology was observed in the hippocampus of both tauWT and tauT175D expressing rats and tauT175D pathology was increased in the presence of cholinergic neuronal expression of human TDP-43M337V. These data indicate that co-expression of pathological TDP-43 and tau protein exacerbate the pathology associated with either individual protein.

Stress granules (SGs) are phase-separated, membraneless, cytoplasmic ribonucleoprotein (RNP) assemblies whose primary function is to promote cell survival by condensing translationally stalled mRNAs, ribosomal components, translation initiation factors, and RNA-binding proteins (RBPs). While the protein composition and the function of proteins in the compartmentalization and the dynamics of assembly and disassembly of SGs has been a matter of study for several years, the role of RNA in these structures had remained largely unknown. RNA species are, however, not passive members of RNA granules in that RNA by itself can form homo and heterotypic interactions with other RNA molecules leading to phase separation and nucleation of RNA granules. RNA can also function as molecular scaffolds recruiting multivalent RBPs and their interactors to form higher-order structures. With the development of SG purification techniques coupled to RNA-seq, the transcriptomic landscape of SGs is becoming increasingly understood, revealing the enormous potential of RNA to guide the assembly and disassembly of these transient organelles. SGs are not only formed under acute stress conditions but also in response to different diseases such as viral infections, cancer, and neurodegeneration. Importantly, these granules are increasingly being recognized as potential precursors of pathological aggregates in neurodegenerative diseases. In this review, we examine the current evidence in support of RNA playing a significant role in the formation of SGs and explore the concept of SGs as therapeutic targets.

There is increasing acceptance that amyotrophic lateral sclerosis (ALS), classically considered a neurodegenerative disease affecting almost exclusively motor neurons, is syndromic with both clinical and biological heterogeneity. This is most evident in its association with a broad range of neuropsychological, behavioral, speech and language deficits [collectively termed ALS frontotemporal spectrum disorder (ALS-FTSD)]. Although the most consistent pathology of ALS and ALS-FTSD is a disturbance in TAR DNA binding protein 43 kDa (TDP-43) metabolism, alterations in microtubule-associated tau protein (tau) metabolism can also be observed in ALS-FTSD, most prominently as pathological phosphorylation at Thr175 (pThr175tau). pThr175 has been shown to promote exposure of the phosphatase activating domain (PAD) in the tau N-terminus with the consequent activation of GSK3β mediated phosphorylation at Thr231 (pThr231tau) leading to pathological oligomer formation. This pathological cascade of tau phosphorylation has been observed in chronic traumatic encephalopathy with ALS (CTE-ALS) and in both in vivo and in vitro experimental paradigms, suggesting that it is of critical relevance to the pathobiology of ALS-FTSD. It is also evident that the co-existence of alterations in the metabolism of TDP-43 and tau acts synergistically in a rodent model to exacerbate the pathology of either.

Abstract Neurofilaments (NFs) are the most abundant cytoskeletal component of vertebrate myelinated axons. NFs function by determining axonal caliber, promoting axonal growth and forming a 3-dimensional lattice that supports the organization of cytoplasmic organelles. The stoichiometry of NF protein subunits (NFL, NFM and NFH) has to be tightly controlled to avoid the formation of NF neuronal cytoplasmic inclusions (NCIs), axonal degeneration and neuronal death, all pathological hallmarks of amyotrophic lateral sclerosis (ALS). The post-transcriptional control of NF transcripts is critical for regulating normal levels of NF proteins. Previously, we showed that miRNAs that are dysregulated in ALS spinal cord regulate the levels of NEFL mRNA. In order to complete the understanding of altered NF expression in ALS, in this study we have investigated the regulation of NEFM and NEFH mRNA levels by miRNAs. We observed that a small group of ALS-linked miRNAs that are expressed in human spinal motor neurons directly regulate NEFM and NEFH transcript levels in a manner that is associated with an increase in NFM and NFH protein levels in ALS spinal cord homogenates. In concert with previous observations demonstrating the suppression of NEFL mRNA steady state levels in ALS, these observations provide support for the hypothesis that the dysregulation of miRNAs in spinal motor neurons in ALS fundamentally alters the stoichiometry of NF expression, leading to the formation of pathological NCIs.

Abstract Common pathological features of amyotrophic lateral sclerosis (ALS) include cytoplasmic aggregation of several RNA-binding proteins. Out of these RNA-binding proteins, TDP-43, FUS/TLS and RGNEF have been shown to co-aggregate with one another within motor neurons of sporadic ALS (sALS) patients, suggesting that there may be a common regulatory network disrupted. MiRNAs have been a recent focus in ALS research as they have been identified to be globally down-regulated in the spinal cord of ALS patients. The objective of this study was to identify if there are miRNA(s) dysregulated in sALS that are responsible for regulating the TDP-43, FUS/TLS and RGNEF network. In this study, we identify miR-194 and miR-b2122 to be significantly down-regulated in sALS patients, and were predicted to regulate TARDBP, FUS/TLS and RGNEF expression. Reporter gene assays and RT-qPCR revealed that miR-b2122 down-regulates the reporter gene through direct interactions with either the TARDBP, FUS/TLS, or RGNEF 3’UTR, while miR-194 down-regulates firefly expression when it contained either the TARDBP or FUS/TLS 3’UTR. Further, we showed that miR-b2122 regulates endogenous expression of all three of these genes in a neuronal-derived cell line. Also, an ALS-associated mutation in the FUS/TLS 3’UTR ablates the ability of miR-b2122 to regulate reporter gene linked to FUS/TLS 3’UTR, and sALS samples which showed a down-regulation in miR-b2122 also showed an increase in FUS/TLS protein expression. Overall, we have identified a novel miRNA that is down-regulated in sALS that appears to be a central regulator of disease-related RNA-binding proteins, and thus its dysregulation likely contributes to TDP-43, FUS/TLS and RGNEF pathogenesis in sALS.

Background Physicians are typically appointed to leadership roles within health care organizations on the basis of individual accomplishments in research, education, and/or clinical care. However, these types of achievements seldom provide the requisite management capabilities to lead within health organizations. In this manuscript, we described our initial experience in developing an in-house program to provide current and aspiring physician leaders with the managerial capabilities to enhance the quality of health care delivery within their respective organization. Methods In a partnership established between a Medical School and a Business School, we designed two series of weekend workshops to provide current and aspiring physician leaders with the financial capabilities to assist them in their future healthcare leadership careers. This course was then expanded to a Management Principles for Physician workshop with open enrollment to physicians at all levels. Baseline demographics and participant evaluations of each course were recorded. In the open enrollment Management Principles for Physician workshop, we examined the relationship between participant background and their course evaluations as well as their areas of interest for further training. Results All 3 workshops received excellent evaluations by participants. The positive impact of the open enrollment program, based on participants' self-evaluations, was the highest in female physicians, as well as early to mid-career physicians. Additionally, physicians who do not currently hold leadership positions and those who are leading at Divisional levels were the most interested in further training in finance. Conclusion In summary, this series of workshops demonstrated the feasibility of an in-house physician leadership program and yielded important information for the design of future leadership development curriculum.

Approximately 50–60% of all patients with amyotrophic lateral sclerosis (ALS) will develop a deficit of frontotemporal function, ranging from frontotemporal dementia (FTD) to one or more deficits of neuropsychological, speech or language function which are collectively known as the frontotemporal spectrum disorders of ALS (ALS-FTSD). While the neuropathology underlying these disorders is most consistent with a widespread alteration in the metabolism of transactive response DNA-binding protein 43 (TDP-43), in both ALS with cognitive impairment (ALSci) and ALS with FTD (ALS-FTD; also known as MND-FTD) there is evidence for alterations in the metabolism of the microtubule associated protein tau. This alteration in tau metabolism is characterized by pathological phosphorylation at residue Thr175 (pThr175 tau) which in vitro is associated with activation of GSK3β (pTyr216GSK3β), phosphorylation of Thr231tau, and the formation of cytoplasmic inclusions with increased rates of cell death. This putative pathway of pThr175 induction of pThr231 and the formation of pathogenic tau inclusions has been recently shown to span a broad range of tauopathies, including chronic traumatic encephalopathy (CTE) and CTE in association with ALS (CTE-ALS). This pathway can be experimentally triggered through a moderate traumatic brain injury, suggesting that it is a primary neuropathological event and not secondary to a more widespread neuronal dysfunction. In this review, we discuss the neuropathological underpinnings of the postulate that ALS is associated with a tauopathy which manifests as a FTSD, and examine possible mechanisms by which phosphorylation at Thr175tau is induced. We hypothesize that this might lead to an unfolding of the hairpin structure of tau, activation of GSK3β and pathological tau fibril formation through the induction of cis-Thr231 tau conformers. A potential role of TDP-43 acting synergistically with pathological tau metabolism is proposed.

MiRNAs are key regulators of the mammalian transcriptome that have been increasingly linked to degenerative diseases of the motor neurons. Although many of the miRNAs currently incriminated as participants in the pathogenesis of these diseases are also important to the normal development and function of motor neurons, at present there is no knowledge of the complete miRNA profile of motor neurons. In this review, we examine the current understanding with respect to miRNAs that are specifically required for motor neuron development, function and viability, and provide evidence that these should be considered as a functional network of miRNAs which we have collectively termed MotomiRs. We will also summarize those MotomiRs currently known to be associated with both amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), and discuss their potential use as biomarkers.

Amyotrophic lateral sclerosis (ALS) is considered a multifactorial, multisystem disease in which inflammation and the immune system play important roles in development and progression. The pleiotropic cytokine TNFα is one of the major players governing the inflammation in the central nervous system and peripheral districts such as the neuromuscular and immune system. Changes in TNFα levels are reported in blood, cerebrospinal fluid, and nerve tissues of ALS patients and animal models. However, whether they play a detrimental or protective role on the disease progression is still not clear. Our group and others have recently reported opposite involvements of TNFR1 and TNFR2 in motor neuron death. TNFR2 mediates TNFα toxic effects on these neurons presumably through the activation of MAP kinase-related pathways. On the other hand, TNFR2 regulates the function and proliferation of regulatory T cells (Treg) whose expression is inversely correlated with the disease progression rate in ALS patients. In addition, TNFα is considered a procachectic factor with a direct catabolic effect on skeletal muscles, causing wasting. We review and discuss the role of TNFα in ALS in the light of its multisystem nature.

Amyotrophic lateral sclerosis (ALS) is a fatal disease of unknown etiology. Mutations in copper/zinc superoxide dismutase (SOD1) are the most commonly associated genetic abnormality. Given that SOD1 is ubiquitously expressed, the exclusive vulnerability of motor neurons is one of the most puzzling issues in ALS research. We here report that wild-type SOD1 mRNA forms ribonucleoprotein (RNP) complexes with protein homogenates of neuronal tissue but not with homogenates of non-neuronal tissues. 3′ Untranslated region of SOD1 mRNA-dependent RNP complexes functioned to stabilize SOD1 mRNA. Moreover, SOD1 mRNAs harboring ALS-associated mutations, including silent mutations, were deficient in forming RNP complexes. In contrast, SOD1 mRNAs harboring artificial mutations, not known to be associated with ALS, demonstrated preserved RNP complex formation. This paper reports RNP complex formation on SOD1 mRNA as a neuronal tissue-specific and ALS-associated mutation sensitive feature.

Roughly 400,000 people in the U.S. are living with bone metastases, the vast majority occurring in the spine. Metastases to the spine result in fractures, pain, paralysis, and significant health care costs. This predilection for cancer to metastasize to the bone is seen across most cancer histologies, with the greatest incidence seen in prostate, breast, and lung cancer. The molecular process involved in this predilection for axial versus appendicular skeleton is not fully understood, although it is likely that a combination of tumor and local micro-environmental factors plays a role. Immune cells are an important constituent of the bone marrow microenvironment and many of these cells have been shown to play a significant role in tumor growth and progression in soft tissue and bone disease. With this in mind, we sought to examine the differences in immune landscape between axial and appendicular bones in the normal noncancerous setting in order to obtain an understanding of these landscapes. To accomplish this, we utilized mass cytometry by time-of-flight (CyTOF) to examine differences in the immune cell landscapes between the long bone and vertebral body bone marrow from patient clinical samples and C57BL/6J mice. We demonstrate significant differences between immune populations in both murine and human marrow with a predominance of myeloid progenitor cells in the spine. Additionally, cytokine analysis revealed differences in concentrations favoring a more myeloid enriched population of cells in the vertebral body bone marrow. These differences could have clinical implications with respect to the distribution and permissive growth of bone metastases.