Background: Mitochondrial dysfunction is a critical therapeutic target in amyotrophic lateral sclerosis (ALS). Oxaloacetate (OAA) is a promising candidate therapy as it crosses the blood brain barrier, reaches motor neurons, and enhances mitochondrial bioenergetics with positive preclinical data in ALS. Methods: We conducted a prospective, phase 1B, dose escalation study using a standard 3+3 design to assess the safety profile and determine the maximum tolerated dose. Dose-limiting toxicity (DLT) was defined as any serious adverse event (SAE) requiring hospitalization or any adverse event (AE) attributed to OAA that required discontinuation of the medication. Dosages evaluated started at 1000 mg twice daily in Cohort 1 and if tolerated were escalated by 500mg up to 2500 mg twice daily in the maximal dose Cohort 4. To determine target engagement, we evaluated a panel of mitochondrial biomarkers, platelet TDP-43 levels, and MR spectroscopy of brain glutathione from baseline and at end of treatment. Results: A total of 19 participants were screened, 18 enrolled, and one patient at the 2500mg BID dose withdrew due to a DLT. OAA was overall well tolerated up to a dose of 2500 mg BID. Among the small sample of participants, no consistent signal of target engagement was observed, although in aggregate post-exposure MRS determined brain glutathione levels increased. Conclusions: This study supports the safety and tolerability of OAA at doses up to 2500 mg BID in patients with ALS. A future trial would be warranted to confirm maximum tolerated dose, to assess efficacy and further explore target engagement.

Diabetes is associated with systemic bioenergetic dysfunction that contributes to complications such as diabetic peripheral neuropathy (DPN). While mitochondrial impairment has been implicated, the role of non-mitochondrial pathways is less clearly defined. To address this gap, we optimized platelet-based extracellular flux assays as a minimally invasive platform to study human bioenergetics. A seeding density of 20 × 106 platelets per well provided reliable respiratory measurements, consistent with the presence of 5–8 mitochondria per platelet. Platelets from both type 2 diabetes (T2D) and DPN patients demonstrated a significant reduction in non-mitochondrial oxygen consumption rate (OCR) compared with controls. This finding suggests impairment of oxygen-consuming processes beyond the mitochondria, likely reflecting diminished activity of oxidoreductase enzymes such as NADPH oxidases, cyclooxygenases, and lipoxygenases, which regulate redox balance, inflammatory signaling, and vascular tone. In contrast, basal and ATP-linked OCR were only marginally reduced in T2D and not significantly altered in DPN, indicating that mitochondrial-linked dysfunction may be more subtle or heterogeneous in these patient populations. The consistent decrease in non-mitochondrial OCR across both T2D and DPN highlights its potential as an early and sensitive indicator of systemic bioenergetic dysregulation in diabetes. These findings demonstrate that platelets are a practical and repeatable resource for assessing bioenergetics in humans. Diabetes impairs both mitochondrial and non-mitochondrial respiration, with the latter showing the most robust alterations. Reduced non-mitochondrial OCR may contribute independently to the pathogenesis of DPN and holds promise as a biomarker for prognosis and therapeutic monitoring in diabetic complications. ### Competing Interest Statement The authors have declared no competing interest. Kansas City Area Life Sciences Institute, https://ror.org/01sp6c076, KCALSI 17-8

Optic atrophy 1(OPA1) is a GTPase protein that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In neurodegenerative diseases such as Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), the mitochondrial network morphology is compromised. Studies on TAR-DNA binding protein 43 (TDP-43) has been the focus in our lab. OPA1 and TDP-43 interaction may shed a light on how aberrant TDP-43 interacts with OPA1, which will lead to mitochondrial dysfunction. The preliminary study tested the idea of whether OPA1 and TDP-43 are physically interacting in human platelet derived mitochondria obtained from healthy human subjects.

Introduction Phenylbutyrate (PBA) showed positive effect on the muscle cell model of Inclusion Body Myositis (IBM) by improving lysosomal activity, ameliorating consequences of impaired autophagy, and decreasing vacuolization. This provides rationale to study this medication in patients with IBM. Objectives To evaluate the safety and tolerability of phenylbutyrate in IBM, and monitor for any early signal of effectiveness. Methods Open-label study of 10 subjects with IBM who received treatment with PBA for 3 months after a 3-month run-in period. The PBA dose was 3 gm twice daily. The primary outcome measure was adverse event reporting. Secondary outcome measures included manual muscle testing, timed up and go test, IBM functional rating scale, and grip strength, along with exploratory biomarkers evaluating the mitochondrial function, stress response, degenerative process, and apoptosis. Results Ten subjects completed the study. PBA was well tolerated with no serious adverse events related to it. The most common adverse events were gastrointestinal related and did not require stopping treatment. One of the biomarkers (MitoTracker) showed a statistically significant drop over the treatment period of the study (p-value of 0.02 for the mean change). There were no statistically significant changes in other secondary outcome measures, but the study was limited by a small sample size and short treatment period. Conclusions Phenylbutyrate was safe and well tolerated in patients with IBM in this pilot study. The change in the MitoTracker suggests target engagement, but a Phase II study is needed to confirm and study the efficacy of PBA in IBM

The objective of this study is to assess in Inclusion Body Myositis (IBM) the safety of an autologous graft consisting of adipose-derived regenerative cells (ADRCs) derived from the Celution 800/CRS System.

The TAR DNA Binding Protein 43 (TDP-43) has been implicated in the pathogenesis of human neurodegenerative diseases and exhibits hallmark neuropathology in amyotrophic lateral sclerosis (ALS). Here, we explore its tractability as a plasma biomarker of disease and describe its localization and possible functions in the cytosol of platelets. Novel TDP-43 immunoassays were developed on three different technical platforms and qualified for specificity, signal-to-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples. Surprisingly, implementation of these assays demonstrated that biobank-archived plasma samples yielded considerable heterogeneity in TDP-43 levels. Importantly, subsequent investigation attributed these differences to variable platelet recovery. Fractionations of fresh blood revealed that >= 95% of the TDP-43 in platelet-containing plasma was compartmentalized within the platelet cytosol. We reasoned that this highly concentrated source of TDP-43 comprised an interesting substrate for biochemical analyses. Additional characterization of platelets revealed the presence of the disease-associated phosphoserine 409/410 TDP-43 proteoform and many neuron- and astrocyte-expressed TDP-43 mRNA targets. Considering these striking similarities, we propose that TDP-43 may serve analogous functional roles in platelets and synapses, and that the study of platelet TDP-43 might provide a window into disease-related TDP-43 dyshomeostasis in the central nervous system.

The assignment of blood-based biomarkers for neurodegenerative diseases is of great clinical value. Well-developed and validated blood-based biomarkers can serve in early diagnosis and prognosis as well as aid in patient screening when recruiting for clinical trials. We attempted to establish a portfolio for post-translationally modified TAR DNA/RNA-binding protein (TDP-43), a regulator of nuclear transcription, in platelet cytosol obtained from patients with Alzheimer’s disease (AD) comparing to age-matched healthy subjects and a disease control cohort. We aimed to identify the most prominent post-translational modifications of TDP-43 as an AD-relevant biomarker and to demonstrate that such an assessment can be performed in peripheral blood. We have isolated TDP-43 protein from human platelet cytosol utilizing an Immunoaffinity chromatography. The eluates were immunoprobed with a series of antibodies raised against post-translationally modified proteins. We employed a capillary electrophoretic immunoassay (CEI) to assess the phosphorylated TDP-43 profile. We observed that SUMOylation, phosphorylation, ubiquitination, and cysteine oxidation of TDP-43 are more prominent in platelet cytosol of AD patients as compared to control subjects. These studies will pave the way for identifying disease-specific TDP-43 derivatives that can be potential biomarkers for early diagnosis and the development of therapeutics.

The mitochondrial electron transfer complex (ETC) profile is modified in the heart tissue of the offspring born to an exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis.

Amyotrophic lateral sclerosis (ALS) remains a devastating motor neuron disease with limited treatment options. Oxaloacetate treatment has a neuroprotective effect in rodent models of seizure and neurodegeneration. Therefore, we treated the ALS model superoxide dismutase 1 (SOD1) G93A mice with oxaloacetate and evaluated their neuromuscular function and lifespan. Treatment with oxaloacetate beginning in the presymptomatic stage significantly improved neuromuscular strength measured during the symptomatic stage in the injected mice compared to the non-treated group. Oxaloacetate treatment starting in the symptomatic stage significantly delayed limb paralysis compared with the non-treated group. For lifespan analysis, oxaloacetate treatment did not show a statistically significant positive effect, but the treatment did not shorten the lifespan. Mechanistically, SOD1G93A mice showed increased levels of tumor necrosis factor-α (TNFα) and peroxisome proliferative activated receptor gamma coactivator 1α (PGC-1α) mRNAs in the spinal cord. However, oxaloacetate treatment reverted these abnormal levels to that of wild-type mice. Similarly, the altered expression level of total NF-κB protein returned to that of wild-type mice with oxaloacetate treatment. These results suggest that the beneficial effects of oxaloacetate treatment in SOD1G93A mice may reflect the effects on neuroinflammation or bioenergetic stress.

Objective Blood-based biomarkers provide a crucial information in progress of neurodegenerative diseases with minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in central nervous system. Methods Exosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analysed by Western blotting method. Results The profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compare to control subjects. Conclusions Serum-derived exosomal biomolecules can serve as surrogate biomarkers in neuro degenerative diseases. Key Messages A canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis. Serum-derived exosomes contains Transactive Response DNA Binding Protein 43 (TDP-43), potential biomarker candidate. The levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited a similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.

Early detection of amyotrophic lateral sclerosis (ALS) is critical for better therapeutic outcomes. The median time from symptom onset to diagnosis of ALS is 11 months, with a range of 6-21 months. Given that the median life expectancy is three years, it is important to shorten the diagnostic journey, initiate therapies promptly, and facilitate clinical research participation. Biomarkers may be the key to enhancing early diagnosis, tracking disease progression, and testing target engagement of promising therapeutics. Clinically valid biomarkers for ALS are currently lacking, and research has been ongoing to identify appropriate biomarkers. Ideal biomarkers should be minimally invasive, such as blood. In this chapter, we review our current understanding of blood based biomarker research in ALS and discuss future directions.

Blood-based biomarkers are much-needed diagnostic and prognostic tools for ALS. Canine degenerative myelopathy (DM) is recognized animal disease model to study the biology of human ALS. Serum derived exosomes are potential carrier that transport intercellular hormone-like messengers, together with their stability as carrier of proteins and RNA, make them ideal as biomarkers for a variety of diseases and biological processes. We study exosomal TDP-43 pattern as a surrogate biomarker that reflects biochemical changes in central nervous system. We isolated exosomes from canine serum using commercial exosome isolation reagents. TDP-43 and SOD1 profile in spinal cord homogenate lysate and that of serum-derived exosomes were found elevated in dogs with DM. We conclude levels of spinal cord TDP-43 and serum-derived exomes were similar in TDP-43 profiling, which warrant further investigation of disease sensitivity and specificity for establishing as a blood-based biomarker in canine DM.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%-20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Measurement of the electron transfer cascade (ETC) enzyme activities and their kinetic profiles is important in assessing mitochondrial function in the nervous system in health and disease or following exposure to toxic agents. The optimization of enzymatic assays for brain tissues and neurons is critical to the development of high-throughput assay formats. This article describes a step-by-step protocol for reliable and reproducible assessment of ETC enzyme kinetics (Complex I-IV) for mitochondria from small quantities of tissue from different brain regions, such as the hippocampus, cerebellum, and frontal cortex, or from neurons in culture. Methods for differential and density gradient centrifugation are detailed for isolating cell body and synaptic mitochondria from brain, as well as measurement of ETC activities in microwell plate or single-cuvette format using spectrophotometric methods. Easy-to follow assay layouts and useful tips are presented, allowing the user to perform these assays in under 3 hr. © 2019 by John Wiley & Sons, Inc.

Aim Platelets provide substantial information about the proteolytic system profile in neurodegenerative diseases. Assessment of autophagy and proteasome target proteins in platelets may reflect tissue proteolytic machinery profile in central nervous system in Alzheimer’s diseases (AD). We aimed to demonstrate the optimum assay conditions and identify target proteins in platelet proteolytic machinery. Methods Platelet samples were obtained from clinically verified AD patients and age-matched non-demented control subjects that were recruited by University of Kansas Alzheimer’s disease Center. Autophagosome participating proteins in platelets were identified by Western blotting analysis. Standard gel electrophoresis and electro transfer apparatus were used for protein transfer onto the membrane. Several antibodies were tested to identify the best working antibodies, and their concentrations were optimized. An ELISA kit was used for platelet proteasome protein determination. Infrared imaging technology was used for visualizing the proteins on the membrane. Results Autophagosome participating proteins showed elevated levels in AD patient platelet cytosol. Only LC3-I autophagosome protein levels were significantly elevated. The concentrations of platelet lysate proteasome were assessed. AD patient’s proteasome levels were elevated but they were statistically not important as compared to controls. Conclusions Platelets can be used for assessing whether proteolytic system is functional. Blood-based sampling from human donors is less-invasive and analyzing platelet proteolytic system profile may help to develop pharmaceutical intervention approaches for neurodegenerative diseases in general.

Protein aggregation trends in neurodegenerative diseases are largely unmapped due to the complex nature of protein-protein interactions and their regulatory machineries such as protein proteolytic systems. Since the protein aggregation process in humans is a slow process, early determination of the patients that will develop neurodegenerative diseases later in life is critical in terms of starting effec-tive treatment, which will reduce the expensive health care. In this chapter, I will discuss the nature of protein aggregation of signature proteins and the status of protein proteolytic systems such as proteasome and autophagosome in Alzheimer ’ s disease, Parkinson ’ s disease, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Huntington ’ s disease, and prion disease under the light of recent studies including our new findings.

Impaired interactions between Calcineurin (Cn) and (Cu/Zn) superoxide dismutase (SOD1) are suspected to be responsible for the formation of hyperphosphorylated protein aggregation in amyotrophic lateral sclerosis (ALS). Serine (Ser)- enriched phosphorylated TDP-43 protein aggregation appears in the spinal cord of ALS animal models, and may be linked to the reduced phosphatase activity of Cn. The mutant overexpressed SOD1G93A protein does not properly bind zinc (Zn) in animal models; hence, mutant SOD1G93A-Cn interaction weakens. Consequently, unstable Cn fails to dephosphorylate TDP-43 that yields hyperphosphorylated TDP-43 aggregates. Our previous studies had suggested that Cn and SOD1 interaction was necessary to keep Cn enzyme functional. We have observed low Cn level, increased Zn concentrations, and increased TDP-43 protein levels in cervical, thoracic, lumbar, and sacral regions of the spinal cord tissue homogenates. This study further supports our previously published work indicating that Cn stability depends on functional Cn-SOD1 interaction because Zn is crucial for maintaining the Cn stability. Less active Cn did not efficiently dephosphorylate TDP-43; hence TDP-43 aggregations appeared in the spinal cord tissue.

Introduction: Rasagiline is a monoamine oxidase B (MAO-B) inhibitor with possible neuroprotective effects in patients with amyotrophic lateral sclerosis (ALS). Methods: We performed a randomized, double-blind, placebo-controlled trial of 80 ALS participants with enrichment of the placebo group with historical controls (n = 177) at 10 centers in the United States. Participants were randomized in a 3:1 ratio to 2 mg/day rasagiline or placebo. The primary outcome was average slope of decline on the ALS Functional Rating Scale-Revised (ALSFRS-R). Secondary measures included slow vital capacity, survival, mitochondrial and molecular biomarkers, and adverse-event reporting. Results: There was no difference in the average 12-month ALSFRS-R slope between rasagiline and the mixed placebo and historical control cohorts. Rasagiline did not show signs of drug-target engagement in urine and blood biomarkers. Rasagiline was well tolerated with no serious adverse events. Discussion: Rasagiline did not alter disease progression compared with controls over 12 months of treatment. Muscle Nerve 59:201-207, 2019

Aim: Alzheimer’s disease (AD) and other forms of dementia create a non-curable disease population in World’s societies. To develop a blood-based biomarker is important so that the remedial or disease-altering therapeutic intervention for AD patients would be available at the early stage. Materials & Methods: TDP-43 levels were analyzed in post-mortem brain tissue and platelets of AD and control subjects. Results: We observed an increased TDP-43 (<60%) in post-mortem AD brain regions and similar trends were also observed in patient’s platelets. Conclusion: Platelet TDP-43 could be used as a surrogate biomarker that is measurable, reproducible, and sensitive for screening the patients with some early clinical signs of AD and can be used to monitor disease prognosis. Lay abstract In this study, we explore to identify an Alzheimer’s disease-selective phospho-specific antibody that recognizes the diseased form of TDP-43 protein in patient’s blood-derived platelets. Our results suggest that selective anti-phosphorylated TDP-43 antibody discriminates Alzheimer’s disease from non-demented controls and patients with amyotrophic lateral sclerosis. Therefore, platelet screening with a selective antibody could potentially be a useful tool for diagnostic purposes for Alzheimer’s disease.