Biology subjects

Milbrandt, J.

Publications and source records attributed to Milbrandt, J..

At least 19 recordsLinked to original sources

bioRxiv2026

Mitochondrial NAD+ import gates neuronal resilience to active SARM1

SARM1 is an inducible NADase that executes pathological axon degeneration, but how NAD+ destruction causes neuronal failure remains unclear. Because SARM1 is predicted to act outside the mitochondrial matrix, cytosolic NAD+ loss has been viewed as the critical lethal lesion. We show that SARM1 activation rapidly depletes cytosolic and mitochondrial NAD+ and collapses ATP in both compartments, revealing early, coupled metabolic failure. Enhancing mitochondrial NAD+ import through SLC25A51, the mitochondrial NAD+ transporter, preserves mitochondrial NAD+, maintains mitochondrial function, and delays axon degeneration and neuronal death despite ongoing SARM1 activity. Unexpectedly, SLC25A51 restores cytosolic NAD+-dependent metabolism, reversing a glycolytic block and increasing NAD+ synthesis during stress. Glycolytic inhibition attenuates protection, identifying cytosolic metabolic recovery as an important component of neuronal resilience. AAV-SLC25A51 improves function in a chronic SARM1-dependent neuropathy model. Thus, mitochondrial NAD+ import gates SARM1-driven degeneration by limiting coupled mitochondrial-cytosolic metabolic failure, presenting a downstream intervention point for axon preservation.

neuroscience↗

bioRxiv2025

Dysregulated lactate metabolism synergizes with ALS genetic risk factors to accelerate motor decline

Neurons rely on glial lactate shuttling for metabolic support, which declines with aging and in neurodegenerative disease. Full disruption of lactate shuttling in peripheral nerves causes progressive axon degeneration, but we were interested to understand how partial disruption, a scenario more relevant to aging and disease, contributes to neurodegeneration risk. Pyruvate and lactate are interconverted by lactate dehydrogenases (LDHA and LDHB) in both lactate producing and consuming cells. We therefore began by investigating Ldhb knockout mice (loss of LDHA, the dominant LDH in liver and muscle, caused embryonic lethality), and discovered that they develop progressive neuromuscular junction atrophy and functional decline without axon degeneration. Because even Ldhb+/- heterozygosity significantly affects motor behavior, we also wondered about a potential link to congenital disease and pursued this by identifying rare loss-of-function LDHB variants among ALS patients. Next, to better understand how LDHB loss leads to motor decline, we selectively deleted it in defined cell types. SC-specific deletion caused robust motor defects, whereas motor neuron-specific deletion has little effect. Reasoning that neuronal LDHB deficiency could model age-associated decline in lactate metabolism, we asked whether it would interact with ALS genetic risk. Indeed, motor-neuron LDHB deficiency synergizes with relatively mild ALS risk variants-- TDP43Q331Kand Sod1D83G knock-in alleles--to produce early motor neuropathy, indicating that LDHB loss enhances disease risk. These findings establish lactate metabolism as a modifier of motor system vulnerability and highlight it as a therapeutic target in peripheral as well as central neurodegeneration.

neuroscience↗

bioRxiv2025

TDP-43 suppression of ATP8A2 cryptic splicing implicates phosphatidylserine-driven neuroinflammation in ALS/FTD

Inappropriate externalization of phosphatidylserine (PS) is a candidate mechanism of pathogenic neuroinflammation, a critical driver of neurodegenerative disease. ATP8A2, a flippase that maintains PS on the plasma membrane inner leaflet, is mutated in both Wabbler-lethal mice and patients with the ataxia syndrome CAMRQ4. Here, we identify ATP8A2 as a target of TDP-43 cryptic exon suppression, and demonstrate that ATP8A2 loss leads to immune-mediated neurodegeneration. ATP8A2 splicing is significantly dysregulated following TDP-43 depletion in human neurons and in brains of patients with Amyotrophic Lateral Sclerosis-Frontotemporal Dementia (ALS-FTD). In mice, Atp8a2 loss increases PS exposure and promotes neuroinflammation. Depletion of peripheral macrophages rescues motor axon degeneration and doubles Atp8a2 knockout mouse lifespan, while depletion of both peripheral macrophages and central microglia quadruples lifespan and improves coordination. Hence, ATP8A2 is a pathologically relevant TDP-43 target and inhibition of phagocytic immune cell attack against neurons is a potential treatment for patients with CAMRQ4 and ALS-FTD.

neuroscience↗

bioRxiv2025

SARM1 is an essential component of neuronal Parthanatos

The NAD+ hydrolase SARM1 is the central executioner of pathological axon degeneration. SARM1 is allosterically activated by an increased NMN/NAD+ ratio resulting from depletion of NAD+ or accumulation of its precursor, NMN, typically due to loss of the labile NAD+ synthetase NMNAT2 following axon injury. Another NAD+ hydrolase, PARP1, is hyperactivated by DNA damage, triggering the Parthanatos cell death pathway. We demonstrate that multiple mechanistically-distinct DNA-damaging agents lead to SARM1 activation and axon degeneration following PARP1 activation. Remarkably, SARM1 is required for key steps downstream of PARP1 activation by DNA damage that are pathognomonic of Parthanatos, including mitochondrial depolarization, nuclear translocation of AIF (apoptosis-inducing factor), and cell death. Moreover, SARM1 mediates glutamate excitotoxicity, a clinically significant pathomechanism attributed to Parthanatos. The identification of SARM1 as an essential component of neuronal Parthanatos, a major contributor to cell death in neurodegenerative disease, greatly expands the potential clinical utility of SARM1 inhibitors.

neuroscience↗

bioRxiv2024

Suppressing phagocyte activation by overexpressing the phosphatidylserine lipase ABHD12 preserves sarmopathic nerves

Programmed axon degeneration (AxD) is a key feature of many neurodegenerative diseases. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of AxD, preventing it from initiating the rapid local NAD+ depletion and metabolic catastrophe that precipitates axon destruction. Because these components of the AxD pathway act within neurons, it was also assumed that the timetable of AxD was set strictly by a cell-intrinsic mechanism independent of neuron-extrinsic processes later activated by axon fragmentation. However, using a rare human disease model of neuropathy caused by hypomorphic NMNAT2 mutations and chronic SARM1 activation (sarmopathy), we demonstrated that neuronal SARM1 can initiate macrophage-mediated axon elimination long before stressed-but-viable axons would otherwise succumb to cell-intrinsic metabolic failure. Investigating potential SARM1-dependent signals that mediate macrophage recognition and/or engulfment of stressed-but-viable axons, we found that chronic SARM1 activation triggers axonal blebbing and dysregulation of phosphatidylserine (PS), a potent phagocyte immunomodulatory molecule. Neuronal expression of the phosphatidylserine lipase ABDH12 suppresses nerve macrophage activation, preserves motor axon integrity, and rescues motor function in this chronic sarmopathy model. We conclude that PS dysregulation is an early SARM1-dependent axonal stress signal, and that blockade of phagocytic recognition and engulfment of stressed-but-viable axons could be an attractive therapeutic target for management of neurological disorders involving SARM1 activation.

neuroscience↗

bioRxiv2024

Reduced STMN2 and pathogenic TDP-43, two hallmarks of ALS, synergize to accelerate motor decline in mice

Pathological TDP-43 loss from the nucleus and cytoplasmic aggregation occurs in almost all cases of ALS and half of frontotemporal dementia patients. Stathmin2 (Stmn2) is a key target of TDP-43 regulation and aberrantly spliced Stmn2 mRNA is found in patients with ALS, frontotemporal dementia, and Alzheimers Disease. STMN2 participates in the axon injury response and its depletion in vivo partially replicates ALS-like symptoms including progressive motor deficits and distal NMJ denervation. The interaction between STMN2 loss and TDP-43 dysfunction has not been studied in mice because TDP-43 regulates human but not murine Stmn2 splicing. Therefore, we generated trans-heterozygous mice that lack one functional copy of Stmn2 and express one mutant TDP-43Q331K knock-in allele to investigate whether reduced STMN2 function exacerbates TDP-43-dependent pathology. Indeed, we observe synergy between these two alleles, resulting in an early onset, progressive motor deficit. Surprisingly, this behavioral defect is not accompanied by detectable neuropathology in the brain, spinal cord, peripheral nerves or at neuromuscular junctions (NMJs). However, the trans-heterozygous mice exhibit abnormal mitochondrial morphology in their distal axons and NMJs. As both STMN2 and TDP-43 affect mitochondrial dynamics, and neuronal mitochondrial dysfunction is a cardinal feature of many neurodegenerative diseases, this abnormality likely contributes to the observed motor deficit. These findings demonstrate that partial loss of STMN2 significantly exacerbates TDP-43-associated phenotypes, suggesting that STMN2 restoration could ameliorate TDP-43 related disease before the onset of degeneration.

neuroscience↗

bioRxiv2023

The Cytoskeletal Stress Response Pathway: a homeostatic system driven by Dual Leucine Zipper Kinase (DLK)

Genetic and pharmacological perturbation of the cytoskeleton enhances the regenerative potential of neurons. This response requires Dual-leucine Zipper Kinase (DLK), a neuronal stress sensor that is a central regulator of axon regeneration and degeneration. The damage and repair aspects of this response are reminiscent of other cellular homeostatic systems, suggesting that a cytoskeletal homeostatic response exists. In this study, we propose a framework for understanding DLK mediated neuronal cytoskeletal homeostasis. We demonstrate that a) low dose nocodazole treatment activates DLK signaling and b) DLK signaling mitigates the microtubule damage caused by the cytoskeletal perturbation. We also perform RNA-seq to discover a DLK-dependent transcriptional signature. This signature includes genes likely to attenuate DLK signaling while simultaneously inducing actin regulating genes and promoting actin-based morphological changes to the axon. These results are consistent with the model that cytoskeletal disruption in the neuron induces a DLK-dependent homeostatic mechanism, which we term the Cytoskeletal Stress Response (CSR) pathway.

neuroscience↗

bioRxiv2023

The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and Interleukin-1/18 receptor resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using HDX-MS. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using 2pFLIM which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIREA protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

biochemistry↗

bioRxiv2022

Disruption of lactate metabolism in the peripheral nervous system leads to motor-selective deficits

Schwann cells (SCs) myelinate and provide trophic support to axons in the peripheral nervous system (PNS) and disruption of SC cellular metabolism leads to demyelination and axon degeneration, both symptoms of peripheral neuropathies. The lactate shuttle hypothesis proposes that glycolytic support cells supply lactate to adjacent axons to sustain their high metabolic demands, a process that requires the interconversion of lactate and pyruvate via lactate dehydrogenase (LDH) in both SCs and neurons. To test this hypothesis in the PNS, we selectively knocked out the genes for both LDH enzymes, LDHA and LDHB, in motor neurons (MNs), sensory neurons (SNs), or SCs. Interestingly, motor axons and their synapses progressively degenerate when LDH is deleted from either MNs or SCs; however, defects in sensory axons or their terminals were not observed when LDH was excised from either SNs or SCs. Deletion of LDH in SCs also leads to a decrease in total ATP levels in peripheral nerves despite a marked accumulation of pyruvate and glycolytic intermediates, consistent with the failure of pyruvate to lactate conversion in SCs leading to energetic deficits in axons. These results support a model in which motor axons are more dependent on SC-derived lactate than are sensory axons, a specific dependency that suggests LDH and lactate shuttling influence the course of motor-dominated neuropathies such as ALS.

neuroscience↗

bioRxiv2022

SARM1 promotes axonal, synaptic, and mitochondrial pathologies in Charcot-Marie-Tooth disease type 2A

Charcot-Marie-Tooth disease (CMT) type 2A is an axonal neuropathy caused by mutations in the mitofusin 2 (MFN2) gene. MFN2 mutations result in profound mitochondrial abnormalities, but the mechanism underlying axonal pathology is unknown. SARM1, the central executioner of axon degeneration, can induce neuropathy and is activated by dysfunctional mitochondria. We tested the role of SARM1 in a rat model carrying a dominant CMT2A mutation (Mfn2H361Y) that exhibits progressive dying-back axonal degeneration, NMJ abnormalities, muscle atrophy, and mitochondrial abnormalities, all hallmarks of the human disease. We generated Sarm1 knockout and Mfn2H361Y, Sarm1 double mutant rats and find that deletion of SARM1 rescues axonal, synaptic, and muscle phenotypes, demonstrating that SARM1 induces much of the neuropathology in this model. Despite the presence of mutant Mfn2 protein in these double mutant rats, loss of SARM1 also dramatically suppressed many mitochondrial defects, including the number, size, and cristae density defects of synaptic mitochondria. This surprising finding indicates that dysfunctional mitochondria activate SARM1, and activated SARM1 feeds back on mitochondria to exacerbate mitochondrial pathology. As such, this work identifies SARM1 inhibition as an exciting therapeutic candidate for the treatment of CMT2A and other neurodegenerative diseases with prominent mitochondrial pathology.

neuroscience↗

bioRxiv2022

Chemical structures of cyclic ADP ribose (cADPR) isomers and the molecular basis of their production and signaling

Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD+ hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2cADPR) and v2-cADPR (3cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression. One-Sentence SummaryThe chemical structures of two O-glycosidic bond-containing cyclic ADP ribose isomers, the molecular basis of their production, and their function in antiviral and plant immunity suppression by bacteria are reported.

biochemistry↗

bioRxiv2022

Loss of Stathmin-2, a hallmark of TDP-43-associated ALS, causes motor neuropathy

TDP-43 mediates proper Stathmin-2 (STMN2) mRNA splicing, and STMN2 protein is reduced in the spinal cord of most ALS patients. To test the hypothesis that STMN2 loss contributes to ALS pathogenesis, we generated constitutive and conditional STMN2 knockout mice. Constitutive STMN2 loss results in early-onset sensory and motor neuropathy featuring impaired motor behavior and dramatic distal neuromuscular junction (NMJ) denervation of fast-fatigable motor units, which are selectively vulnerable in ALS, without axon or motoneuron degeneration. Selective excision of STMN2 in motoneurons leads to similar NMJ pathology. STMN2 KO heterozygous mice, which better model the partial loss of STMN2 protein found in ALS patients, display a slowly progressive, motor-selective neuropathy with functional deficits and NMJ denervation. Thus, our findings strongly support the hypothesis that STMN2 reduction due to TDP-43 pathology contributes to ALS pathogenesis.

neuroscience↗

bioRxiv2022

Macrophage depletion blocks congenital SARM1-dependent neuropathy

Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified two rare NMNAT2 missense variants in two brothers afflicted with a progressive neuropathy syndrome. The polymorphisms result in amino acid substitutions, V98M and R232Q, which reduce NMNAT2 NAD+-synthetase activity. We generated a mouse model of the human syndrome and found that Nmnat2V98M/Nmnat2R232Q compound-heterozygous CRISPR mice survive to adulthood but develop progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes are all SARM1-dependent. Remarkably, macrophage depletion therapy blocks and reverses neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induces an inflammatory neuropathy and highlight the potential of immune therapy to treat this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

neuroscience↗

bioRxiv2021

Defining hierarchical protein interaction networks from spectral analysis of bacterial proteomes

Cellular phenotypes emerge from a hierarchy of molecular interactions: proteins interact to form complexes, pathways, and phenotypes. We show that hierarchical networks of protein interactions can be extracted from the statistical pattern of proteome variation as measured across thousands of bacteria and that these hierarchies reflect the emergence of complex bacterial phenotypes. We describe the mathematics underlying our statistical approach and validate our results through gene-set enrichment analysis and comparison to existing experimentally-derived hierarchical databases. We demonstrate the biological utility of our unbiased hierarchical models by creating a model of motility in Pseudomonas aeruginosa and using it to discover a previously unappreciated genetic effector of twitch-based motility. Overall, our approach, SCALES (Spectral Correlation Analysis of Layered Evolutionary Signals), predicts hierarchies of protein interaction networks describing emergent biological function using only the statistical pattern of bacterial proteome variation.

systems biology↗

bioRxiv2021

Nicotinic acid mononucleotide is an allosteric SARM1 inhibitor promoting axonal protection

SARM1 is an inducible NAD+ hydrolase that is the central executioner of pathological axon loss. Recently, we elucidated the molecular mechanism of SARM1 activation, demonstrating that SARM1 is a metabolic sensor regulated by the levels of NAD+ and its precursor, nicotinamide mononucleotide (NMN), via their competitive binding to an allosteric site within the SARM1 N-terminal ARM domain. In healthy neurons with abundant NAD+, binding of NAD+ blocks access of NMN to this allosteric site. However, with injury or disease the levels of the NAD+ biosynthetic enzyme NMNAT2 drop, increasing the NMN/ NAD+ ratio and thereby promoting NMN binding to the SARM1 allosteric site, which in turn induces a conformational change activating the SARM1 NAD+ hydrolase. Hence, NAD+ metabolites both regulate the activation of SARM1 and, in turn, are regulated by the SARM1 NAD+ hydrolase. This dual upstream and downstream role for NAD+ metabolites in SARM1 function has hindered mechanistic understanding of axoprotective mechanisms that manipulate the NAD+ metabolome. Here we reevaluate two methods that potently block axon degeneration via modulation of NAD+ related metabolites, 1) the administration of the NMN biosynthesis inhibitor FK866 in conjunction with the NAD+ precursor nicotinic acid riboside (NaR) and 2) the neuronal expression of the bacterial enzyme NMN deamidase. We find that these approaches not only lead to a decrease in the levels of the SARM1 activator NMN, but also an increase in the levels of the NAD+ precursor nicotinic acid mononucleotide (NaMN). We show that NaMN inhibits SARM1 activation, and demonstrate that this NaMN-mediated inhibition is important for the long-term axon protection induced by these treatments. Analysis of the NaMN-ARM domain co-crystal structure shows that NaMN competes with NMN for binding to the SARM1 allosteric site and promotes the open, autoinhibited configuration of SARM1 ARM domain. Together, these results demonstrate that the SARM1 allosteric pocket can bind a diverse set of metabolites including NMN, NAD+, and NaMN to monitor cellular NAD+ homeostasis and regulate SARM1 NAD+ hydrolase activity. The relative promiscuity of the allosteric site may enable the development of potent pharmacological inhibitors of SARM1 activation for the treatment of neurodegenerative disorders. HighlightsO_LINaMN binds to SARM1 N-terminal allosteric site and inhibits SARM1 NAD+ hydrolase C_LIO_LINaMN inhibits SARM1 activation by stabilizing its open, inactive structure C_LIO_LINMN deamidase promotes strong axonal protection by reducing NMN and increasing NaMN C_LI

neuroscience↗

bioRxiv2021

Neurotoxins subvert the allosteric activation mechanism of SARM1 to induce neuronal loss

SARM1 is an inducible TIR-domain NAD+ hydrolase that mediates pathological axon degeneration. SARM1 is activated by an increased ratio of NMN to NAD+, which competes for binding to an allosteric activating site. When NMN binds, the TIR domain is released from autoinhibition, activating its NAD+ hydrolase activity. The discovery of this allosteric activating site led us to hypothesize that other NAD+-related metabolites might also activate SARM1. Here we show that the nicotinamide analogue 3-acetylpyridine (3-AP), first identified as a neurotoxin in the 1940s, is converted to 3-APMN which activates SARM1 and induces SARM1-dependent NAD+ depletion, axon degeneration and neuronal death. Systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to peripheral nerve induces SARM1-dependent axon degeneration. We also identify a related pyridine derivative, 2-aminopyridine, as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity, and furthermore, suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

neuroscience↗

bioRxiv2021

Live Imaging Reveals the Cellular Events Downstream of SARM1 Activation

SARM1 is an inducible NAD+ hydrolase that triggers axon loss and neuronal cell death in the injured and diseased nervous system. While SARM1 activation and enzyme function are well defined, the cellular events downstream of SARM1 activity but prior to axonal demise are much less well understood. Defects in calcium, mitochondria, ATP, and membrane homeostasis occur in injured axons, but the relationships among these events have been difficult to disentangle because prior studies analyzed large collections of axons in which cellular events occur asynchronously. Here we used live imaging with single axon resolution to investigate the cellular events downstream of SARM1 activity. Our studies support a model in which SARM1 NADase activity leads to an ordered sequence of events from loss of cellular ATP, to defects in mitochondrial movement and depolarization, followed by calcium influx, externalization of phosphatidylserine, and loss of membrane permeability prior to catastrophic axonal self-destruction.

neuroscience↗

bioRxiv2021

Constitutively active SARM1 variants found in ALS patients induce neuropathy

In response to injury, neurons activate a program of organized axon self-destruction initiated by the NAD+ hydrolase SARM1. In healthy neurons SARM1 is autoinhibited, but single amino acid changes can abolish autoinhibition leading to constitutively-active SARM1 enzymes that promote degeneration when expressed in cultured neurons. To investigate whether naturally-occurring human variants might similarly disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of 29 rare SARM1 alleles identified among 8,507 amyotrophic lateral sclerosis (ALS) patients. Ten missense variants or small in-frame deletions exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. Expression of these constitutively active ALS-associated SARM1 alleles in cultured dorsal root ganglion (DRG) neurons is pro-degenerative and cytotoxic. Intrathecal injection of an AAV expressing the common SARM1 reference allele is innocuous to mice, but a construct harboring SARM1V184G, the constitutively active variant found most frequently in the ALS patients, causes axon loss, motor dysfunction, and sustained neuroinflammation. These results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions.

neuroscience↗