Assessment of blood-brain barrier leakage and brain oxygenation in Connexin-32 knockout mice with systemic neuroinflammation using pulse electron paramagnetic resonance imaging techniques.

PURPOSE: The determination of blood-brain barrier (BBB) integrity and partial pressure of oxygen (pO2) in the brain is of substantial interest in several neurological applications. This study aimed to assess the feasibility of using trityl OX071-based pulse electron paramagnetic resonance imaging (pEPRI) to provide a quantitative estimate of BBB integrity and pO2 maps in mouse brains as a function of neuroinflammatory disease progression. METHODS: Five Connexin-32 (Cx32)-knockout (KO) mice were injected with lipopolysaccharide to induce neuroinflammation for imaging. Three wild-type mice were also used to optimize the imaging procedure and as control animals. An additional seven Cx32-KO mice were used to establish the BBB leakage of trityl using the colorimetric assay. All pEPRI experiments were performed using a preclinical instrument, JIVA-25 (25 mT/720 MHz), at times t = 0, 4, and 6 h following lipopolysaccharide injection. Two pEPRI imaging techniques were used: (a) single-point imaging for obtaining spatial maps to outline the brain and calculate BBB leakage using the signal amplitude, and (b) inversion-recovery electron spin echo for obtaining pO2 maps. RESULTS: A statistically significant change in BBB leakage was found using pEPRI with the progression of inflammation in Cx32 KO animals. However, the change in pO2 values with the progression of inflammation for these animals was not statistically significant. CONCLUSIONS: For the first time, we show the ability of pEPRI to provide pO2 maps in mouse brains noninvasively, along with a quantitative assessment of BBB leakage. We expect this study to open new queries from the field to explore the pathology of many neurological diseases and provide a path to new treatments.

Mechanisms of Diseases Associated with Mutation in GJC2/Connexin 47.

Connexins are members of a family of integral membrane proteins that provide a pathway for both electrical and metabolic coupling between cells. Astroglia express connexin 30 (Cx30)-GJB6 and Cx43-GJA1, while oligodendroglia express Cx29/Cx31.3-GJC3, Cx32-GJB1, and Cx47-GJC2. Connexins organize into hexameric hemichannels (homomeric if all subunits are identical or heteromeric if one or more differs). Hemichannels from one cell then form cell-cell channels with a hemichannel from an apposed cell. (These are termed homotypic if the hemichannels are identical and heterotypic if the hemichannels differ). Oligodendrocytes couple to each other through Cx32/Cx32 or Cx47/Cx47 homotypic channels and they couple to astrocytes via Cx32/Cx30 or Cx47/Cx43 heterotypic channels. Astrocytes couple via Cx30/Cx30 and Cx43/Cx43 homotypic channels. Though Cx32 and Cx47 may be expressed in the same cells, all available data suggest that Cx32 and Cx47 cannot interact heteromerically. Animal models wherein one or in some cases two different CNS glial connexins have been deleted have helped to clarify the role of these molecules in CNS function. Mutations in a number of different CNS glial connexin genes cause human disease. Mutations in GJC2 lead to three distinct phenotypes, Pelizaeus Merzbacher like disease, hereditary spastic paraparesis (SPG44) and subclinical leukodystrophy.

New ASH initiatives to improve patient care in the long-overlooked sickle cell disease.

Because of the unique biology of sickle cell disease (SCD) as well as the societal disadvantages and racial inequities suffered by these patients, individuals with SCD have not benefited from the same remarkable advances in care and therapeutics as those with other hematologic disorders. Life expectancy of individuals with SCD is shortened by ∼20 years even with optimal clinical care, and infant mortality continues to be a major concern in low-income countries. As hematologists, we must do more. The American Society of Hematology (ASH) and the ASH Research Collaborative have instituted a multipronged initiative to improve the lives of individuals living with this disease. Here, we describe 2 components of this ASH initiative, the Consortium on Newborn Screening in Africa (CONSA) to improve the early diagnosis of infants in low-resource countries and the SCD Clinical Trial Network to accelerate the development of more effective therapeutics and care for those with this disorder. The combination of SCD-focused initiatives, ASH Research Collaborative, CONSA, and Sickle Cell Clinical Trials Network has enormous potential to dramatically alter the course of SCD worldwide. We believe that the timing is ripe to embark on these critical and worthwhile initiatives and improve the lives of individuals with this disease.

Individual phosphatidylinositol transfer proteins have distinct functions that do not involve lipid transfer activity.

Platelets use signal transduction pathways facilitated by class I phosphatidylinositol transfer proteins (PITPs). The 2 mammalian class I PITPs, PITPα and PITPß, are single PITP domain soluble proteins that are encoded by different genes and share 77% sequence identity, although their individual roles in mammalian biology remain uncharacterized. These proteins are believed to shuttle phosphatidylinositol and phosphatidylcholine between separate intracellular membrane compartments, thereby regulating phosphoinositide synthesis and second messenger formation. Previously, we observed that platelet-specific deletion of PITPα, the predominantly expressed murine PITP isoform, had no effect on hemostasis but impaired tumor metastasis formation and disrupted phosphoinositide signaling. Here, we found that mice lacking the less expressed PITPß in their platelets exhibited a similar phenotype. However, in contrast to PITPα-null platelet lysates, which have impaired lipid transfer activity, PITPß-null platelet lysates have essentially normal lipid transfer activity, although both isoforms contribute to phosphoinositide synthesis in vitro. Moreover, we found that platelet-specific deletion of both PITPs led to ex vivo platelet aggregation/secretion and spreading defects, impaired tail bleeding, and profound tumor dissemination. Our study also demonstrated that PITP isoforms are required to maintain endogenous phosphoinositide PtdInsP2 levels and agonist-stimulated second messenger formation. The data shown here demonstrate that the 2 isoforms are functionally overlapping and that a single isoform is able to maintain the homeostasis of platelets. However, both class I PITP isoforms contribute to phosphoinositide signaling in platelets through distinct biochemical mechanisms or different subcellular domains.

Pharmacologic Targeting of the C-Terminus of Heat Shock Protein 90 Improves Neuromuscular Function in Animal Models of Charcot Marie Tooth X1 Disease.

Charcot-Marie-Tooth X1 (CMTX1) disease is an inherited peripheral neuropathy that arises from loss-of-function mutations in the protein connexin 32 (Cx32). CMTX1 currently lacks a pharmacologic approach toward disease management, and we have previously shown that modulating the expression of molecular chaperones using novologue therapy may provide a viable disease-modifying approach to treat metabolic and demyelinating neuropathies. Cemdomespib is an orally bioavailable novologue that manifests neuroprotective activity by modulating the expression of heat shock protein 70 (Hsp70). We examined if 1 to 5 months of daily cemdomespib therapy may improve neuropathic symptoms in three mouse models of CMTX1 (Cx32 deficient (Cx32def), T55I-Cx32def, and R75W-Cx32 mice). Daily drug therapy significantly improved motor nerve conduction velocity (MNCV) and grip strength in all three models, but the compound muscle action potential was only improved in Cx32def mice. Drug efficacy required Hsp70 as improvements in MNCV, and the grip strength was abrogated in Cx32def × Hsp70 knockout mice. Five months of novologue therapy was associated with improved neuromuscular junction morphology, femoral motor nerve myelination, reduction in foamy macrophages, and a decrease in Schwann cell c-jun levels. To determine if c-jun may be downstream of Hsp70 and necessary for drug efficacy, c-jun expression was specifically deleted in Schwann cells of Cx32def mice. While the deletion of c-jun worsened the neuropathy, cemdomespib therapy remained effective in improving MNCV and grip strength. Our data show that cemdomespib therapy improves CMTX1-linked neuropathy in an Hsp70-dependent but a c-jun-independent manner and without regard to the nature of the underlying Cx32 mutation.

Targeting pleckstrin-2/Akt signaling reduces proliferation in myeloproliferative neoplasm models.

Myeloproliferative neoplasms (MPNs) are characterized by the activated JAK2/STAT pathway. Pleckstrin-2 (Plek2) is a downstream target of the JAK2/STAT5 pathway and is overexpressed in patients with MPNs. We previously revealed that Plek2 plays critical roles in the pathogenesis of JAK2-mutated MPNs. The nonessential roles of Plek2 under physiologic conditions make it an ideal target for MPN therapy. Here, we identified first-in-class Plek2 inhibitors through an in silico high-throughput screening approach and cell-based assays, followed by the synthesis of analogs. Plek2-specific small-molecule inhibitors showed potent inhibitory effects on cell proliferation. Mechanistically, Plek2 interacts with and enhances the activity of Akt through the recruitment of downstream effector proteins. The Plek2-signaling complex also includes Hsp72, which protects Akt from degradation. These functions were blocked by Plek2 inhibitors via their direct binding to the Plek2 dishevelled, Egl-10 and pleckstrin (DEP) domain. The role of Plek2 in activating Akt signaling was further confirmed in vivo using a hematopoietic-specific Pten-knockout mouse model. We next tested Plek2 inhibitors alone or in combination with an Akt inhibitor in various MPN mouse models, which showed significant therapeutic efficacies similar to that seen with the genetic depletion of Plek2. The Plek2 inhibitor was also effective in reducing proliferation of CD34-positive cells from MPN patients. Our studies reveal a Plek2/Akt complex that drives cell proliferation and can be targeted by a class of antiproliferative compounds for MPN therapy.

Knock-in mouse models for CMTX1 show a loss of function phenotype in the peripheral nervous system.

The X-linked form of Charcot-Marie-Tooth disease (CMTX1) is the second most common form of CMT. In this study we used CRISPR/Cas9 to develop new "knock-in" models of CMTX1 that are more representative of the spectrum of mutations seen with CMTX1 than the Cx32 knockout (KO) mouse model used previously. We compared mice of four genotypes - wild-type, Cx32KO, p.T55I, and p.R75W. Sciatic motor conduction velocity slowing was the most robust electrophysiologic indicator of neuropathy, showing reductions in the Cx32KO by 3 months and in the p.T55I and p.R75W mice by 6 months. At both 6 and 12 months, all three mutant genotypes showed reduced four limb and hind limb grip strength compared to WT mice. Performance on 6 and 12 mm width balance beams revealed deficits that were most pronounced at on the 6 mm balance beam at 6 months of age. There were pathological changes of myelinated axons in the femoral motor nerve in all three mutant lines by 3 months of age, and these became more pronounced at 6 and 12 months of age; sensory nerves (femoral sensory and the caudal nerve of the tail) appeared normal at all ages examined. Our results demonstrate that mice can be used to show the pathogenicity of human GJB1 mutations, and these new models for CMTX1 should facilitate the preclinical work for developing treatments for CMTX1.

SARS-CoV-2 Vaccination-Induced Thrombotic Thrombocytopenia: A Rare but Serious Immunologic Complication.

Billions of individuals worldwide have benefited from the unprecedented large-scale rollout of COVID-19 vaccines. Given the sheer number of people that have received these vaccines, it is not surprising that rare side effects are reported that were not previously detected in the phase III vaccine trials. This review addresses one rare complication called SARS-CoV-2 vaccination-induced thrombotic thrombocytopenia (VITT). It occurs in approximately 1/50,000 to 1/100,000 recipients of the adenovirus vector-based COVID-19 vaccines made by AstraZeneca-Oxford or Johnson & Johnson. Information on VITT syndrome was disseminated quickly via social media and publications after it was first discovered. Initial observations associating VITT with specific patient populations, thrombus locations, and outcomes associated with heparin therapy have since been refined with additional clinical experience. In this review, we discuss what is currently known about the incidence, pathophysiology, diagnosis, and treatment of VITT.

Genotype-phenotype correlations and disease mechanisms in PEX13-related Zellweger spectrum disorders.

BACKGROUND: Pathogenic variants in PEX-genes can affect peroxisome assembly and function and cause Zellweger spectrum disorders (ZSDs), characterized by variable phenotypes in terms of disease severity, age of onset and clinical presentations. So far, defects in at least 15 PEX-genes have been implicated in Mendelian diseases, but in some of the ultra-rare ZSD subtypes genotype-phenotype correlations and disease mechanisms remain elusive. METHODS: We report five families carrying biallelic variants in PEX13. The identified variants were initially evaluated by using a combination of computational approaches. Immunofluorescence and complementation studies on patient-derived fibroblasts were performed in two patients to investigate the cellular impact of the identified mutations. RESULTS: Three out of five families carried a recurrent p.Arg294Trp non-synonymous variant. Individuals affected with PEX13-related ZSD presented heterogeneous clinical features, including hypotonia, developmental regression, hearing/vision impairment, progressive spasticity and brain leukodystrophy. Computational predictions highlighted the involvement of the Arg294 residue in PEX13 homodimerization, and the analysis of blind docking predicted that the p.Arg294Trp variant alters the formation of dimers, impairing the stability of the PEX13/PEX14 translocation module. Studies on muscle tissues and patient-derived fibroblasts revealed biochemical alterations of mitochondrial function and identified mislocalized mitochondria and a reduced number of peroxisomes with abnormal PEX13 concentration. CONCLUSIONS: This study expands the phenotypic and mutational spectrum of PEX13-related ZSDs and also highlight a variety of disease mechanisms contributing to PEX13-related clinical phenotypes, including the emerging contribution of secondary mitochondrial dysfunction to the pathophysiology of ZSDs.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Activation of the unfolded protein response by Connexin47 mutations associated with Pelizaeus-Merzbacher-like disease.

Pelizaeus-Merzbacher-like disease type 1 (PMLD1) is a hypomyelinating disorder arising in patients with mutations in GJC2, encoding Connexin47 (Cx47). PMLD1 causes nystagmus, cerebellar ataxia, spasticity and changes in CNS white matter detected by MRI. At least one mutation (p.I33M) yields a much milder phenotype, spastic paraplegia type 44 (SPG44). Cx47 contributes to gap junction communication channels between oligodendrocytes (OLs), the myelinating cells in the central nervous system (CNS), and between OLs and astrocytes. Prior studies in cell lines have shown that PMLD1 mutants such as p.P87S display defective protein trafficking, intracellular retention in the ER and loss-of-function. Here we show that when expressed in primary OLs, three PMLD1 associated mutants (p.P87S, p.Y269D and p.M283T) show ER retention of Cx47 and evidence of activation of the cellular stress (unfolded protein response, UPR) and apoptotic pathways. On the other hand, the milder SPG44 associated mutation p.I33M shows a wild-type-like subcellular distribution and no activation of the UPR or apoptotic pathways. These studies provide new insight into a potential element of toxic gain of function underlying the mechanism of PMLD1 that should help guide future therapeutic approaches.

AAV1.NT-3 gene therapy for X-linked Charcot-Marie-Tooth neuropathy type 1.

X-linked Charcot-Marie-Tooth neuropathy (CMTX) is caused by mutations in the gene encoding Gap Junction Protein Beta-1 (GJB1)/Connexin32 (Cx32) in Schwann cells. Neurotrophin-3 (NT-3) is an important autocrine factor supporting Schwann cell survival and differentiation and stimulating axon regeneration and myelination. Improvements in these parameters have been shown previously in a CMT1 model, TremblerJ mouse, with NT-3 gene transfer therapy. For this study, scAAV1.tMCK.NT-3 was delivered to the gastrocnemius muscle of 3-month-old Cx32 knockout (KO) mice. Measurable levels of NT-3 were found in the serum at 6-month post gene delivery. The outcome measures included functional, electrophysiological and histological assessments. At 9-months of age, NT-3 treated mice showed no functional decline with normalized compound muscle action potential amplitudes. Myelin thickness and nerve conduction velocity significantly improved compared with untreated cohort. A normalization toward age-matched wildtype histopathological parameters included increased number of Schmidt-Lanterman incisures, and muscle fiber diameter. Collectively, these findings suggest a translational application to CMTX1.

Tmprss6-ASO as a tool for the treatment of Polycythemia Vera mice.

Polycythemia Vera (PV) is a chronic myeloproliferative neoplasm resulting from an acquired driver mutation in the JAK2 gene of hematopoietic stem and progenitor cells resulting in the overproduction of mature erythrocytes and abnormally high hematocrit, in turn leading to thromboembolic complications. Therapeutic phlebotomy is the most common treatment to reduce the hematocrit levels and consequently decrease thromboembolic risk. Here we demonstrate that, by using the iron restrictive properties of the antisense oligonucleotides against Tmprss6 mRNA, we can increase hepcidin to achieve effects equivalent to therapeutic phlebotomy. We provide evidence that this less invasive approach could represent an additional therapeutic tool for the treatment of PV patients.

ASH Research Collaborative: a real-world data infrastructure to support real-world evidence development and learning healthcare systems in hematology.

The ASH Research Collaborative is a nonprofit organization established through the American Society of Hematology's commitment to patients with hematologic conditions and the science that informs clinical care and future therapies. The ASH Research Collaborative houses 2 major initiatives: (1) the Data Hub and (2) the Clinical Trials Network (CTN). The Data Hub is a program for hematologic diseases in which networks of clinical care delivery sites are developed in specific disease areas, with individual patient data contributed through electronic health record (EHR) integration, direct data entry through electronic data capture, and external data sources. Disease-specific data models are constructed so that data can be assembled into analytic datasets and used to enhance clinical care through dashboards and other mechanisms. Initial models have been built in multiple myeloma (MM) and sickle cell disease (SCD) using the Observational Medical Outcomes Partnership (OMOP) Common Data Model (CDM) and Fast Healthcare Interoperability Resources (FHIR) standards. The Data Hub also provides a framework for development of disease-specific learning communities (LC) and testing of health care delivery strategies. The ASH Research Collaborative SCD CTN is a clinical trials accelerator that creates efficiencies in the execution of multicenter clinical trials and has been initially developed for SCD. Both components are operational, with the Data Hub actively aggregating source data and the SCD CTN reviewing study candidates. This manuscript describes processes involved in developing core features of the ASH Research Collaborative to inform the stakeholder community in preparation for expansion to additional disease areas.

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. ONE-SENTENCE SUMMARY: The FcγRIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19.

Pleckstrin-2 is essential for erythropoiesis in ß-thalassemic mice, reducing apoptosis and enhancing enucleation.

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as ß-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in ß-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in ß-thalassemia. We hypothesize that compensatory mechanisms are required in ß-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2-a STAT5-dependent lipid binding protein downstream of erythropoietin-as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in ß-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during ß-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in ß-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in ß-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in ß-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in ß-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.

Investigating oligodendrocyte connexins: Heteromeric interactions between Cx32 and mutant or wild-type forms of Cx47 do not contribute to or modulate gap junction function.

Oligodendrocytes express two gap junction forming connexins, connexin 32 (Cx32) and Cx47; therefore, formation of heteromeric channels containing both Cx47 and Cx32 monomers might occur. Mutations in Cx47 cause both Pelizaeus-Merzbacher-like disease Type 1 (PMLD1) and hereditary spastic paraparesis Type 44 (SPG44) and heteromer formation between these mutants and Cx32 may contribute to the pathogenesis of these disorders. Here, we utilized electrophysiological and antibody-based techniques to examine this possibility. When cells expressing both Cx32 and Cx47 were paired with cells expressing either Cx32 or Cx47, properties were indistinguishable from those produced by cells expressing homotypic Cx32 or Cx47 channels. Similarly, pairing cells expressing both Cx32 and Cx47 with cells expressing Cx30 or Cx43 produced channels indistinguishable from heterotypic Cx32/Cx30 or Cx47/Cx43 channels, respectively. The same assessments were performed on cells expressing Cx32 and four mutant forms of Cx47 (p.I33M associated with SPG44 or p.P87S, p.Y269D or p.M283T associated with PMLD1). None of these mutants showed a functional effect on Cx32. Immunostained cells co-expressing Cx32WT (wild type) and Cx47WT showed a Pearson correlation coefficient close to zero, suggesting that any overlap was due to chance. p.Y269D showed a statistically significant negative correlation with Cx32, suggesting that Cx32 and this mutant overlap less than expected by chance. Co-immunoprecipitation of Cx32 with Cx47WT and mutants show only very low levels of co-immunoprecipitated protein. Overall, our data suggest that interactions between PMLD1 or SPG44 mutants and Cx32 gap junctions do not contribute to the pathogenesis of these disorders.

Effects of early crush on aging wild type and Connexin 32 knockout mice: Evidence for a neuroprotective state in CMT1X mouse nerve.

The long-term sequelae of nerve injury as well as age-related neurodegeneration have been documented in numerous studies, however the role of Cx32 in these processes is not well understood. There is a need for better understanding of the molecular mechanisms that underlie long-term suboptimal nerve function and for approaches to prevent or improve it. In this communication we describe our studies using whole animal electrophysiology to examine the long-term sequelae of sciatic nerve crush in both WT and Cx32KO mice, a model of X-linked Charcot Marie Tooth disease, a subtype of inherited peripheral neuropathies. We present results from electrical nerve recordings done 14 to 27 days and 18 to 20 months after a unilateral sciatic nerve crush performed on 35 to 37-day old mice. Contrary to expectations, we find that whereas crush injury leads to a degradation of WT nerve function relative to uninjured nerves at 18 to 20 months, previously crushed Cx32KO nerves perform at the same level as their uninjured counterparts. Thus, 18 to 20 months after injury, WT nerves perform below the level of normal (uninjured) WT nerves in both motor and sensory nerve function. In contrast, measures of nerve function in Cx32KO mice are degraded for sensory axons but exhibit no additional dysfunction in motor axons. Early nerve injury has no negative electrophysiologic effect on the Cx32 KO motor nerves. Based on our prior demonstration that the transcriptomic profile of uninjured Cx32KO and injured WT sciatic nerves are very similar, the lack of an additional effect of crush on Cx32KO motor nerve parameters suggests that Cx32 knockout may implement a form of neuroprotection that limits the effects of subsequent injury.

PIKfyve Deficiency in Myeloid Cells Impairs Lysosomal Homeostasis in Macrophages and Promotes Systemic Inflammation in Mice.

Macrophages are professional phagocytes that are essential for host defense and tissue homeostasis. Proper membrane trafficking and degradative functions of the endolysosomal system are known to be critical for the function of these cells. We have found that PIKfyve, the kinase that synthesizes the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate, is an essential regulator of lysosomal biogenesis and degradative functions in macrophages. Genetically engineered mice lacking PIKfyve in their myeloid cells (PIKfyvefl/fl LysM-Cre) develop diffuse tissue infiltration of foamy macrophages, hepatosplenomegaly, and systemic inflammation. PIKfyve loss in macrophages causes enlarged endolysosomal compartments and impairs the lysosomal degradative function. Moreover, PIKfyve deficiency increases the cellular levels of lysosomal proteins. Although PIKfyve deficiency reduced the activation of mTORC1 pathway and was associated with increased cleavage of TFEB proteins, this does not translate into transcriptional activation of lysosomal genes, suggesting that PIKfyve modulates the abundance of lysosomal proteins by affecting the degradation of these proteins. Our study shows that PIKfyve modulation of lysosomal degradative activity and protein expression is essential to maintain lysosomal homeostasis in macrophages.

Diseases of connexins expressed in myelinating glia.

Connexins are a family of integral membrane proteins most of which form gap junctions and many of which form hemichannels as well. Mutations in at least 9 of the 21 genes encoding human connexin proteins cause human diseases. Mutations in GJB1 (Cx32), expressed in both Schwann cells and oligodendrocytes, cause both a form of inherited peripheral neuropathy and a variety of CNS symptoms. Mutations in GJC2 (Cx47), expressed in oligodendrocytes cause three disorders: a severe early onset dysmyelinating disorder, Pelizaeus-Merzbacher-Like disease (PMLD1 or HLD2); hereditary spastic paraplegia (SPG44), which has a milder phenotype and later onset; and a subclinical leukodystrophy. The clinical phenotypes and genetics associated with each disorder will be reviewed, focusing on features which may provide clues to pathogenesis. In vitro and animal model data which may shed light on these phenotypes will then be discussed along with recent work which may impact on therapeutic approaches for these disorders.