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 Alzheimer's 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.

Sarm1 knockout prevents type 1 diabetic bone disease in females independent of neuropathy.

Patients with diabetes have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild-type mice also developed diabetic bone disease but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independently of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system.

A SARM1-mitochondrial feedback loop drives neuropathogenesis in a Charcot-Marie-Tooth disease type 2A rat model.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an axonal neuropathy caused by mutations in the mitofusin 2 (MFN2) gene. MFN2 mutations result in profound mitochondrial abnormalities, but the mechanism underlying the axonal pathology is unknown. Sterile α and Toll/IL-1 receptor motif-containing 1 (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, neuromuscular junction (NMJ) abnormalities, muscle atrophy, and mitochondrial abnormalities - all hallmarks of the human disease. We generated Sarm1-KO (Sarm1-/-) and Mfn2H361Y Sarm1 double-mutant rats and found that deletion of Sarm1 rescued axonal, synaptic, muscle, and functional phenotypes, demonstrating that SARM1 was responsible for 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 activated SARM1 and that activated SARM1 fed back on mitochondria to exacerbate the mitochondrial pathology. As such, this work identifies SARM1 inhibition as a therapeutic candidate for the treatment of CMT2A and other neurodegenerative diseases with prominent mitochondrial pathology.

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 2 rare NMNAT2 missense variants in 2 brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model to mirror the human syndrome and found that Nmnat2V98M/R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induced inflammatory neuropathy and highlight the potential of immune therapy as a treatment for this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.

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 patients with amyotrophic lateral sclerosis (ALS). 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 knockout heterozygous mice, which better model the partial loss of STMN2 protein found in patients with ALS, display a slowly progressive, motor-selective neuropathy with functional deficits and NMJ denervation. Thus, our findings strongly support the hypothesis that STMN2 reduction owing to TDP-43 pathology contributes to ALS pathogenesis.

Disentangling glial diversity in peripheral nerves at single-nuclei resolution.

The peripheral nerve contains diverse cell types that support its proper function and maintenance. In this study, we analyzed multiple peripheral nerves using single-nuclei RNA sequencing, which allowed us to circumvent difficulties encountered in analyzing cells with complex morphologies via conventional single-cell methods. The resultant mouse peripheral nerve cell atlas highlights a diversity of cell types, including multiple subtypes of Schwann cells (SCs), immune cells and stromal cells. We identified a distinct myelinating SC subtype that expresses Cldn14, Adamtsl1 and Pmp2 and preferentially ensheathes motor axons. The number of these motor-associated Pmp2+ SCs is reduced in both an amyotrophic lateral sclerosis (ALS) SOD1G93A mouse model and human ALS nerve samples. Our findings reveal the diversity of SCs and other cell types in peripheral nerve and serve as a reference for future studies of nerve biology and disease.

Constitutively active SARM1 variants that induce neuropathy are enriched in ALS patients.

BACKGROUND: 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. METHODS: To investigate whether naturally occurring human variants might disrupt SARM1 autoinhibition and potentially contribute to risk for neurodegenerative disease, we assayed the enzymatic activity of all 42 rare SARM1 alleles identified among 8507 amyotrophic lateral sclerosis (ALS) patients and 9671 controls. We then intrathecally injected mice with virus expressing SARM1 constructs to test the capacity of an ALS-associated constitutively active SARM1 variant to promote neurodegeneration in vivo. RESULTS: Twelve out of 42 SARM1 missense variants or small in-frame deletions assayed exhibit constitutive NADase activity, including more than half of those that are unique to the ALS patients or that occur in multiple patients. There is a > 5-fold enrichment of constitutively active variants among patients compared to controls. Expression of 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 among the ALS patients, causes axon loss, motor dysfunction, and sustained neuroinflammation. CONCLUSIONS: These results implicate rare hypermorphic SARM1 alleles as candidate genetic risk factors for ALS and other neurodegenerative conditions.

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 activate SARM1. Here, we show the nicotinamide analog 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. In mice, systemic treatment with 3-AP causes rapid SARM1-dependent death, while local application to the peripheral nerve induces SARM1-dependent axon degeneration. We identify 2-aminopyridine as another SARM1-dependent neurotoxin. These findings identify SARM1 as a candidate mediator of environmental neurotoxicity and suggest that SARM1 agonists could be developed into selective agents for neurolytic therapy.

Cell-autonomous expression of the acid hydrolase galactocerebrosidase.

Lysosomal storage diseases (LSDs) are typically caused by a deficiency in a soluble acid hydrolase and are characterized by the accumulation of undegraded substrates in the lysosome. Determining the role of specific cell types in the pathogenesis of LSDs is a major challenge due to the secretion and subsequent uptake of lysosomal hydrolases by adjacent cells, often referred to as "cross-correction." Here we create and validate a conditional mouse model for cell-autonomous expression of galactocerebrosidase (GALC), the lysosomal enzyme deficient in Krabbe disease. We show that lysosomal membrane-tethered GALC (GALCLAMP1) retains enzyme activity, is able to cleave galactosylsphingosine, and is unable to cross-correct. Ubiquitous expression of GALCLAMP1 fully rescues the phenotype of the GALC-deficient mouse (Twitcher), and widespread deletion of GALCLAMP1 recapitulates the Twitcher phenotype. We demonstrate the utility of this model by deleting GALCLAMP1 specifically in myelinating Schwann cells in order to characterize the peripheral neuropathy seen in Krabbe disease.

Disrupting insulin signaling in Schwann cells impairs myelination and induces a sensory neuropathy.

Although diabetic mice have been studied for decades, little is known about the cell type specific contributions to diabetic neuropathy (DN). Schwann cells (SCs) myelinate and provide trophic support to peripheral nervous system axons. Altered SC metabolism leads to myelin defects, which can be seen both in inherited and DNs. How SC metabolism is altered in DN is not fully understood, but it is clear that insulin resistance underlies impaired lipid metabolism in many cell types throughout the body via the phosphoinositide 3-kinase/protein kinase b (PKB)/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Here, we created an insulin resistant SC by deleting both insulin receptor (INSR) and insulin-like growth factor receptor 1 (IGF1R), to determine the role of this signaling pathway in development and response to injury in order to understand SC defects in DN. We found that myelin is thinner throughout development and adulthood in INSR/IGF1R Schwann cell specific knock out mice. The nerves of these mutant mice had reduced expression of key genes that mediate fatty acid and cholesterol synthesis due to reduced mTOR-sterol regulatory element-binding protein signaling. In adulthood, these mice show sensory neuropathy phenotypes reminiscent of diabetic mice. Altogether, these data suggest that SCs may play an important role in DN and targeting their metabolism could lead to new therapies for DN.

Gene therapy targeting SARM1 blocks pathological axon degeneration in mice.

Axonal degeneration (AxD) following nerve injury, chemotherapy, and in several neurological disorders is an active process driven by SARM1, an injury-activated NADase. Axons of SARM1-null mice exhibit greatly delayed AxD after transection and in models of neurological disease, suggesting that inhibiting SARM1 is a promising strategy to reduce pathological AxD. Unfortunately, no drugs exist to target SARM1. We, therefore, developed SARM1 dominant-negatives that potently block AxD in cellular models of axotomy and neuropathy. To assess efficacy in vivo, we used adeno-associated virus-mediated expression of the most potent SARM1 dominant-negative and nerve transection as a model of severe AxD. While axons of vehicle-treated mice degenerate rapidly, axons of mice expressing SARM1 dominant-negative can remain intact for >10 d after transection, similar to the protection observed in SARM1-null mice. We thus developed a novel in vivo gene therapeutic to block pathological axon degeneration by inhibiting SARM1, an approach that may be applied clinically to treat manifold neurodegenerative diseases characterized by axon loss.

Schwann cell O-GlcNAcylation promotes peripheral nerve remyelination via attenuation of the AP-1 transcription factor JUN.

Schwann cells (SCs), the glia of the peripheral nervous system, play an essential role in nerve regeneration. Upon nerve injury, SCs are reprogrammed into unique "repair SCs," and these cells remove degenerating axons/myelin debris, promote axonal regrowth, and ultimately remyelinate regenerating axons. The AP-1 transcription factor JUN is promptly induced in SCs upon nerve injury and potently mediates this injury-induced SC plasticity; however, the regulation of these JUN-dependent SC injury responses is unclear. Previously, we produced mice with a SC-specific deletion of O-GlcNAc transferase (OGT). This enzyme catalyzes O-GlcNAcylation, a posttranslational modification that is influenced by the cellular metabolic state. Mice lacking OGT in SCs develop a progressive demyelinating peripheral neuropathy. Here, we investigated the nerve repair process in OGT-SCKO mutant mice and found that the remyelination of regenerating axons is severely impaired. Gene expression profiling of OGT-SCKO SCs revealed that the JUN-dependent SC injury program was elevated in the absence of injury and failed to shut down at the appropriate time after injury. This aberrant JUN activity results in abnormalities in repair SC function and redifferentiation and prevents the timely remyelination. This aberrant nerve injury response is normalized in OGT-SCKO mice with reduced Jun gene dosage in SCs. Mechanistically, OGT O-GlcNAcylates JUN at multiple sites, which then leads to an attenuation of AP-1 transcriptional activity. Together, these results highlight the metabolic oversight of the nerve injury response via the regulation of JUN activity by O-GlcNAcylation, a pathway that could be important in the neuropathy associated with diabetes and aging.

Dysregulation of NAD+ Metabolism Induces a Schwann Cell Dedifferentiation Program.

The Schwann cell (SC) is the major component of the peripheral nervous system (PNS) that provides metabolic and functional support for peripheral axons. The emerging roles of SC mitochondrial function for PNS development and axonal stability indicate the importance of SC metabolism in nerve function and in peripheral neuropathies associated with metabolic disorders. Nicotinamide adenine dinucleotide (NAD+) is a crucial molecule in the regulation of cellular metabolism and redox homeostasis. Here, we investigated the roles of NAD+ metabolism in SC functions in vivo by mutating NAMPT, the rate-limiting enzyme of NAD+ biosynthesis, specifically in SCs. NAMPT SC knock-out male and female mice (NAMPT SCKO mice) had delayed SC maturation in development and developed hypomyelinating peripheral neuropathy without axon degeneration or decreased SC survival. JUN, a master regulator of SC dedifferentiation, is elevated in NAMPT SCKO SCs, suggesting that decreased NAD+ levels cause them to arrest at an immature stage. Nicotinic acid administration rescues the NAD+ decline and reverses the SC maturation defect and the development of peripheral neuropathy, indicating the central role of NAD+ in PNS development. Upon nicotinic acid withdrawal in adulthood, NAMPT SCKO mice showed rapid and severe peripheral neuropathy and activation of ERK/MEK/JUN signaling, which in turn promotes SC dedifferentiation. These data demonstrate the importance of NAD+ metabolism in SC maturation and nerve development and maintenance and suggest that altered SC NAD+ metabolism could underlie neuropathies associated with diabetes and aging.SIGNIFICANCE STATEMENT In this study, we showed that Schwann cell differentiation status is critically dependent on NAD+ homeostasis. Aberrant regulation of NAD+ biosynthesis via NAMPT deletion results in a blockade of Schwann cell maturation during development and severe peripheral neuropathy without significant axon loss. The phenotype can be rescued by supplementation with nicotinic acid; however, withdrawal of nicotinic acid leads to Schwann cell dedifferentiation, myelination defects, and death. These results provide new therapeutic possibilities for peripheral neuropathies associated with NAD+ decline during aging or diabetes.

Abnormal Microglia and Enhanced Inflammation-Related Gene Transcription in Mice with Conditional Deletion of Ctcf in Camk2a-Cre-Expressing Neurons.

CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying 3D chromatin structure. Human mutations in CTCF cause intellectual disability and autistic features. Knocking out Ctcf in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out Ctcf postnatally in glutamatergic forebrain neurons under the control of Camk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+ (Ctcf CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor coordination, and decreased sociability by 4 months of age. Ctcf CKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA from Ctcf CKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically from Ctcf CKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia in Ctcf CKO hippocampus. Finally, we found that microglia in Ctcf CKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm that Ctcf KO in postnatal neurons causes a neurobehavioral phenotype in mice and provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction.SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations in CTCF cause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lacking Ctcf in Camk2a-expressing neurons (Ctcf CKO mice) have spatial learning/memory deficits, impaired fine motor skills, subtly altered social interactions, and decreased dendritic spine density. We demonstrate that Ctcf CKO mice overexpress inflammation-related genes in the brain and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron-microglia interactions are factors in the pathology of CTCF deficiency.

Prevention of vincristine-induced peripheral neuropathy by genetic deletion of SARM1 in mice.

Peripheral polyneuropathy is a common and dose-limiting side effect of many important chemotherapeutic agents. Most such neuropathies are characterized by early axonal degeneration, yet therapies that inhibit this axonal destruction process do not currently exist. Recently, we and others discovered that genetic deletion of SARM1 (sterile alpha and TIR motif containing protein 1) dramatically protects axons from degeneration after axotomy in mice. This finding fuels hope that inhibition of SARM1 or its downstream components can be used therapeutically in patients threatened by axonal loss. However, axon loss in most neuropathies, including chemotherapy-induced peripheral neuropathy, is the result of subacute/chronic processes that may be regulated differently than the acute, one time insult of axotomy. Here we evaluate if genetic deletion of SARM1 decreases axonal degeneration in a mouse model of neuropathy induced by the chemotherapeutic agent vincristine. In wild-type mice, 4 weeks of twice-weekly intraperitoneal injections of 1.5 mg/kg vincristine cause pronounced mechanical and heat hyperalgesia, a significant decrease in tail compound nerve action potential amplitude, loss of intraepidermal nerve fibres and significant degeneration of myelinated axons in both the distal sural nerve and nerves of the toe. Neither the proximal sural nerve nor the motor tibial nerve exhibit axon loss. These findings are consistent with the development of a distal, sensory predominant axonal polyneuropathy that mimics vincristine-induced peripheral polyneuropathy in humans. Using the same regimen of vincristine treatment in SARM1 knockout mice, the development of mechanical and heat hyperalgesia is blocked and the loss in tail compound nerve action potential amplitude is prevented. Moreover, SARM1 knockout mice do not lose unmyelinated fibres in the skin or myelinated axons in the sural nerve and toe after vincristine. Hence, genetic deletion of SARM1 blocks the development of vincristine-induced peripheral polyneuropathy in mice. Our results reveal that subacute/chronic axon loss induced by vincristine occurs via a SARM1 mediated axonal destruction pathway, and that blocking this pathway prevents the development of vincristine-induced peripheral polyneuropathy. These findings, in conjunction with previous studies with axotomy and traumatic brain injury, establish SARM1 as the central determinant of a fundamental axonal degeneration pathway that is activated by diverse insults. We suggest that targeting SARM1 or its downstream effectors may be a viable therapeutic option to prevent vincristine-induced peripheral polyneuropathy and possibly other peripheral polyneuropathies.

Schwann Cell O-GlcNAc Glycosylation Is Required for Myelin Maintenance and Axon Integrity.

UNLABELLED: Schwann cells (SCs), ensheathing glia of the peripheral nervous system, support axonal survival and function. Abnormalities in SC metabolism affect their ability to provide this support and maintain axon integrity. To further interrogate this metabolic influence on axon-glial interactions, we generated OGT-SCKO mice with SC-specific deletion of the metabolic/nutrient sensing protein O-GlcNAc transferase that mediates the O-linked addition of N-acetylglucosamine (GlcNAc) moieties to Ser and Thr residues. The OGT-SCKO mice develop tomaculous demyelinating neuropathy characterized by focal thickenings of the myelin sheath (tomacula), progressive demyelination, axonal loss, and motor and sensory nerve dysfunction. Proteomic analysis identified more than 100 O-GlcNAcylated proteins in rat sciatic nerve, including Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans. PRX lacking O-GlcNAcylation is mislocalized within the myelin sheath of these mutant animals. Furthermore, phenotypes of OGT-SCKO and Prx-deficient mice are very similar, suggesting that metabolic control of PRX O-GlcNAcylation is crucial for myelin maintenance and axonal integrity. SIGNIFICANCE STATEMENT: The nutrient sensing protein O-GlcNAc transferase (OGT) mediates post-translational O-linked N-acetylglucosamine (GlcNAc) modification. Here we find that OGT functions in Schwann cells (SCs) to maintain normal myelin and prevent axonal loss. SC-specific deletion of OGT (OGT-SCKO mice) causes a tomaculous demyelinating neuropathy accompanied with progressive axon degeneration and motor and sensory nerve dysfunction. We also found Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans, is O-GlcNAcylated. Importantly, phenotypes of OGT-SCKO and Prx mutant mice are very similar, implying that compromised PRX function contributes to the neuropathy of OGT-SCKO mice. This study will be useful in understanding how SC metabolism contributes to PNS function and in developing new strategies for treating peripheral neuropathy by targeting SC function.

Aberrant Schwann cell lipid metabolism linked to mitochondrial deficits leads to axon degeneration and neuropathy.

Mitochondrial dysfunction is a common cause of peripheral neuropathy. Much effort has been devoted to examining the role played by neuronal/axonal mitochondria, but how mitochondrial deficits in peripheral nerve glia (Schwann cells [SCs]) contribute to peripheral nerve diseases remains unclear. Here, we investigate a mouse model of peripheral neuropathy secondary to SC mitochondrial dysfunction (Tfam-SCKOs). We show that disruption of SC mitochondria activates a maladaptive integrated stress response (ISR) through the actions of heme-regulated inhibitor (HRI) kinase, and causes a shift in lipid metabolism away from fatty acid synthesis toward oxidation. These alterations in SC lipid metabolism result in depletion of important myelin lipid components as well as in accumulation of acylcarnitines (ACs), an intermediate of fatty acid ß-oxidation. Importantly, we show that ACs are released from SCs and induce axonal degeneration. A maladaptive ISR as well as altered SC lipid metabolism are thus underlying pathological mechanisms in mitochondria-related peripheral neuropathies.

Congenital hypomyelinating neuropathy with lethal conduction failure in mice carrying the Egr2 I268N mutation.

Mouse models of human disease are helpful for understanding the pathogenesis of the disorder and ultimately for testing potential therapeutic agents. Here, we describe the engineering and characterization of a mouse carrying the I268N mutation in Egr2, observed in patients with recessively inherited Charcot-Marie-Tooth (CMT) disease type 4E, which is predicted to alter the ability of Egr2 to interact with the Nab transcriptional coregulatory proteins. Mice homozygous for Egr2(I268N) develop a congenital hypomyelinating neuropathy similar to their human counterparts. Egr2(I268N) is expressed at normal levels in developing nerve but is unable to interact with Nab proteins or to properly activate transcription of target genes critical for proper peripheral myelin development. Interestingly, Egr2(I268N/I268N) mutant mice maintain normal weight and have only mild tremor until 2 weeks after birth, at which point they rapidly develop worsening weakness and uniformly die within several days. Nerve electrophysiology revealed conduction block, and neuromuscular junctions showed marked terminal sprouting similar to that seen in animals with pharmacologically induced blockade of action potentials or neuromuscular transmission. These studies describe a unique animal model of CMT, whereby weakness is due to conduction block or neuromuscular junction failure rather than secondary axon loss and demonstrate that the Egr2-Nab complex is critical for proper peripheral nerve myelination.

Predictors of patient-assessed illness severity in irritable bowel syndrome.

BACKGROUND: Conceptual models suggest that "irritable bowel syndrome (IBS) severity" is a multidimensional outcome that is related to, yet distinct from, health-related quality of life (HRQOL). Existing severity questionnaires are largely based on physician rather than patient-based ratings. Since severity is a patient-centered outcome, it is essential that future instruments are based on patients' self-perceptions of severity. We measured patient-derived predictors of severity in a large cohort of IBS patients. METHODS: We performed a cross-sectional analysis in 755 IBS patients recruited at a university-based center. Subjects completed a bowel symptom questionnaire, SCL-90, and SF-36. The main outcome was patient-assessed "overall severity of gastrointestinal symptoms," as measured on a 0-20 scale (20 = most severe). We first developed a conceptual model of IBS, and then performed bivariate analyses to identify biopsychosocial predictors of severity. We then entered significant predictors into a multivariable model to measure the independent association of each predictor with severity. RESULTS: Six factors predicted severity: (a) abdominal pain rating (P < 0.001); (b) belief that "something serious is wrong with body" (P < 0.001); (c) straining with defecation (P= 0.001); (d) myalgias (P= 0.02); (e) urgency with defecation (P= 0.03); and (f) bloating (P= 0.05). Severity correlated highly with HRQOL in bivariate, but not multivariate, analysis. CONCLUSION: Patient-derived severity in IBS is related to, yet distinct from, generic HRQOL. IBS severity is predicted by abdominal pain, bloating, straining, urgency, myalgias, and disease-related concern. These symptoms fall along both poles of the "brain-gut axis," indicating that a full assessment of patient severity must include a balanced biopsychosocial history.

Tslc1 (nectin-like molecule-2) is essential for spermatozoa motility and male fertility.

The nectin-like molecule-2 (TSLC1) is a cell-cell adhesion molecule expressed in testicular germ cells. To directly examine the role of Tslc1 in male fertility, we generated Tslc1+/- mice that have greater than 90% reduction in Tslc1 expression. Tslc1+/- males exhibited reduced fertility and rarely transmitted the Tslc1 mutant allele, whereas Tslc1+/- females were consistently able to transmit the mutant allele. Histologic and electron microscopic analyses of the testes in Tslc1+/- mice demonstrated disruption of the junctional scaffold between germ cells and Sertoli cells. Reduced Tslc1 expression had no effect on germ cell proliferation or apoptosis. While evidence of normal spermatozoal maturation was supported by Fluorescence Activated Cell Sorting (FACS) analysis, spermatozoa from Tslc1+/- mice demonstrated markedly reduced motility without compromised viability. Collectively, these results establish an essential role for Tslc1 in spermatozoal maturation and motility, distinct from other members of the nectin family.