Presenilin1 familial Alzheimer disease mutants inactivate EFNB1- and BDNF-dependent neuroprotection against excitotoxicity by affecting neuroprotective complexes of N-methyl-d-aspartate receptor.

Excitotoxicity is thought to play key roles in brain neurodegeneration and stroke. Here we show that neuroprotection against excitotoxicity by trophic factors EFNB1 and brain-derived neurotrophic factor (called here factors) requires de novo formation of 'survival complexes' which are factor-stimulated complexes of N-methyl-d-aspartate receptor with factor receptor and presenilin 1. Absence of presenilin 1 reduces the formation of survival complexes and abolishes neuroprotection. EPH receptor B2- and N-methyl-d-aspartate receptor-derived peptides designed to disrupt formation of survival complexes also decrease the factor-stimulated neuroprotection. Strikingly, factor-dependent neuroprotection and levels of the de novo factor-stimulated survival complexes decrease dramatically in neurons expressing presenilin 1 familial Alzheimer disease mutants. Mouse neurons and brains expressing presenilin 1 familial Alzheimer disease mutants contain increased amounts of constitutive presenilin 1-N-methyl-d-aspartate receptor complexes unresponsive to factors. Interestingly, the stability of the familial Alzheimer disease presenilin 1-N-methyl-d-aspartate receptor complexes differs from that of wild type complexes and neurons of mutant-expressing brains are more vulnerable to cerebral ischaemia than neurons of wild type brains. Furthermore, N-methyl-d-aspartate receptor-mediated excitatory post-synaptic currents at CA1 synapses are altered by presenilin 1 familial Alzheimer disease mutants. Importantly, high levels of presenilin 1-N-methyl-d-aspartate receptor complexes are also found in post-mortem brains of Alzheimer disease patients expressing presenilin 1 familial Alzheimer disease mutants. Together, our data identify a novel presenilin 1-dependent neuroprotective mechanism against excitotoxicity and indicate a pathway by which presenilin 1 familial Alzheimer disease mutants decrease factor-depended neuroprotection against excitotoxicity and ischaemia in the absence of Alzheimer disease neuropathological hallmarks which may form downstream of neuronal damage. These findings have implications for the pathogenic effects of familial Alzheimer disease mutants and therapeutic strategies.

Vitamin B12 modulates Parkinson's disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection.

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson's disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive-some with unwanted side effects and unclear clinical outcome-alternative types of LRRK2 inhibitors are lacking. Herein we identify 5'-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.

Unwinding of the Substrate Transmembrane Helix in Intramembrane Proteolysis.

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 310-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 310-helical region of the substrate's TMD. This 310-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 310-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.

Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease.

Crohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10-10) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10-8). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases.

Structural model of the dimeric Parkinson's protein LRRK2 reveals a compact architecture involving distant interdomain contacts.

Leucine-rich repeat kinase 2 (LRRK2) is a large, multidomain protein containing two catalytic domains: a Ras of complex proteins (Roc) G-domain and a kinase domain. Mutations associated with familial and sporadic Parkinson's disease (PD) have been identified in both catalytic domains, as well as in several of its multiple putative regulatory domains. Several of these mutations have been linked to increased kinase activity. Despite the role of LRRK2 in the pathogenesis of PD, little is known about its overall architecture and how PD-linked mutations alter its function and enzymatic activities. Here, we have modeled the 3D structure of dimeric, full-length LRRK2 by combining domain-based homology models with multiple experimental constraints provided by chemical cross-linking combined with mass spectrometry, negative-stain EM, and small-angle X-ray scattering. Our model reveals dimeric LRRK2 has a compact overall architecture with a tight, multidomain organization. Close contacts between the N-terminal ankyrin and C-terminal WD40 domains, and their proximity-together with the LRR domain-to the kinase domain suggest an intramolecular mechanism for LRRK2 kinase activity regulation. Overall, our studies provide, to our knowledge, the first structural framework for understanding the role of the different domains of full-length LRRK2 in the pathogenesis of PD.

A Founder Mutation in VPS11 Causes an Autosomal Recessive Leukoencephalopathy Linked to Autophagic Defects.

Genetic leukoencephalopathies (gLEs) are a group of heterogeneous disorders with white matter abnormalities affecting the central nervous system (CNS). The causative mutation in ~50% of gLEs is unknown. Using whole exome sequencing (WES), we identified homozygosity for a missense variant, VPS11: c.2536T>G (p.C846G), as the genetic cause of a leukoencephalopathy syndrome in five individuals from three unrelated Ashkenazi Jewish (AJ) families. All five patients exhibited highly concordant disease progression characterized by infantile onset leukoencephalopathy with brain white matter abnormalities, severe motor impairment, cortical blindness, intellectual disability, and seizures. The carrier frequency of the VPS11: c.2536T>G variant is 1:250 in the AJ population (n = 2,026). VPS11 protein is a core component of HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) protein complexes involved in membrane trafficking and fusion of the lysosomes and endosomes. The cysteine 846 resides in an evolutionarily conserved cysteine-rich RING-H2 domain in carboxyl terminal regions of VPS11 proteins. Our data shows that the C846G mutation causes aberrant ubiquitination and accelerated turnover of VPS11 protein as well as compromised VPS11-VPS18 complex assembly, suggesting a loss of function in the mutant protein. Reduced VPS11 expression leads to an impaired autophagic activity in human cells. Importantly, zebrafish harboring a vps11 mutation with truncated RING-H2 domain demonstrated a significant reduction in CNS myelination following extensive neuronal death in the hindbrain and midbrain. Thus, our study reveals a defect in VPS11 as the underlying etiology for an autosomal recessive leukoencephalopathy disorder associated with a dysfunctional autophagy-lysosome trafficking pathway.

The vasovagal response of the rat: its relation to the vestibulosympathetic reflex and to Mayer waves.

Vasovagal responses (VVRs) are characterized by transient drops in blood pressure (BP) and heart rate (HR) and increased amplitude of low-frequency oscillations in the Mayer wave frequency range. Typical VVRs were induced in anesthetized, male, Long-Evans rats by sinusoidal galvanic vestibular stimulation (sGVS). VVRs were also produced by single sinusoids that transiently increased BP and HR, by 70-90° nose-up tilts, and by 60° tilts of the gravitoinertial acceleration vector using translation while rotating (TWR). The average power of the BP signal in the Mayer wave range increased substantially when tilts were >70° (0.91 g), i.e., when linear accelerations in the x-z plane were ≥0.9-1.0 g. The standard deviations of the wavelet-filtered BP signals during tilt and TWR overlaid when they were normalized to 1 g. Thus, the amplitudes of the Mayer waves coded the magnitude of the linear acceleration ≥1 g acting on the head and body, and the average power in this frequency range was associated with the generation of VVRs. These data show that VVRs are a natural outcome of stimulation of the vestibulosympathetic reflex and are not a disease. The results also demonstrate the usefulness of the rat as a small animal model for studying human VVRs.

Effect of incision choice on outcomes in primary breast augmentation.

BACKGROUND: Capsular contracture (CC) is the most common complication following primary breast augmentation and one of the most common causes of reoperation. Various studies have suggested certain risk factors, including incision choice. OBJECTIVES: The authors investigate a possible association between the three most common breast augmentation incisions (inframammary, periareolar, and transaxillary) and CC. METHODS: The authors conducted a retrospective chart review of 197 primary breast augmentation patients treated between 2003 and 2009. Significant CC was determined to have occurred if the patient required reoperation for her CC. Patients were excluded if they underwent an augmentation/mastopexy, had previously undergone breast surgery, or received shaped silicone gel implants. CC rates were analyzed on a per-patient basis with Fisher's exact test and on a per-breast basis with the Rao-Scott chi-squared test. RESULTS: One hundred eighty-three patients (336 augmented breasts) were included. Average patient age was 36.5 years. Mean follow-up was 392.6 days. Surgical complications included six breasts with CC (1.8%), three with hematoma (0.9%), and one with an infection (0.3%). Transaxillary incisions produced the highest incidence of contracture (6.4%), followed by periareolar (2.4%) and inframammary (0.5%). There was a statistically-significant difference in the incidence of CC among the three incision sites (P=.03). The increased rate seen with transaxillary incisions versus inframammary incisions was also statistically-significant. No significant association between implant fill material and contracture was found (P=.27). CONCLUSIONS: The risk of CC is significantly higher with transaxillary incisions than with periareolar or inframammary incisions. LEVEL OF EVIDENCE: 4.

Donor-site morbidity and patient satisfaction using a composite nipple graft for unilateral nipple reconstruction in the radiated and nonradiated breast.

BACKGROUND: The number of techniques available for nipple reconstruction underscores the notion that achieving an acceptable result remains a challenge. To better assess the value of a composite nipple graft in unilateral breast reconstruction, a study was performed to evaluate donor-site morbidity and patient satisfaction using a composite nipple graft for unilateral nipple reconstruction following mastectomy and reconstruction in radiated and nonradiated patients. METHODS: A retrospective chart review of all patients who underwent composite nipple reconstruction between October of 1993 and February of 2010 was performed. Medical records were reviewed for outcomes and complications. Each patient was asked to complete a previously validated survey to rate color and projection of both nipples, sensation, and contractility of the donor nipple, and whether she would, in retrospect, have the procedure again. RESULTS: Fifty-nine patients were identified who underwent nipple reconstruction using a composite nipple graft. Thirty-four patients (57.6 percent) responded to the survey. Four surveys were returned due to an invalid address. Average time to breast mound completion was 6 months. Average time to complete nipple reconstruction was 3.6 months after breast mound completion. Ninety-seven percent of the reconstructions were successful. CONCLUSIONS: Composite nipple reconstruction is a useful technique that should be considered in unilateral nipple reconstruction and should be especially considered in patients whose breast has been irradiated, for which flap reconstruction for the nipple can be riskier. Although it is not possible to use in all patients, no other technique provides a nipple reconstruction that can so closely match the contralateral side in color, texture, and overall appearance.

Comparing FK-506 with basic fibroblast growth factor (b-FGF) on the repair of a peripheral nerve defect using an autogenous vein bridge model.

OBJECTIVE: The limited availability of donor sites for nerve grafts and the morbidity associated with their harvesting serve as motivating factors to actively conduct research to find alternatives to the status quo. Experimental and clinical studies have shown that a vein segment used to bridge a peripheral nerve defect leads to a functional nerve repair. Both FK-506 and b-FGF have been reported to enhance peripheral nerve regeneration. This study compared the effects of FK-506 with that of b-FGF on peripheral nerve regeneration in a rat autogenous vein graft conduit model. METHODS: The main trunk of the right sciatic nerve was transected and bridged by an autogenous vein in 30 rats. Small osmotic pumps were placed just proximal to the anastomoses. Groups of 10 rats were assigned to receive saline solution, b-FGF (2,000 units), or FK-506 (0.5 mg/kg/day) via the osmotic pumps for 2 weeks. Sciatic nerve regeneration was evaluated by sensory function, walking track analysis, electrophysiologic studies, and light microscopic evaluation. RESULTS: On post-operative day 90, there was a statistically significant difference (p <.005) in nerve regeneration between the rats who received saline compared with those who received FK-506 or b-FGF. This was determined using sensory function tests, sciatic function index, and electrophysiologic studies. The number of nerve axons, as determined by histological analysis, revealed there were significantly more nerve fibers which were regenerated in both experimental groups (FK-506 and b-FGF) when compared with rats who received saline. There was no statistically significant difference in the number of nerve axons that regenerated in rats injected with FK-506 vs. rats injected with b-FGF. CONCLUSION: FK-506 and b-FGF promote similar nerve regeneration in rats compared with control.

Closure of orocutanous fistula using a pedicled expanded deltopectoral flap.

Orocutaneous fistulas are associated with considerable morbidity. Closures of these fistulas are a challenge to the reconstructing surgeon. The aim of treatment is to provide healthy tissue to repair both the oral and cutaneous defects. The use of an expanded pedicled deltopectoral flap for the closure of an orocutaneous fistula in a patient who has undergone bilateral neck dissection and radiation is reported.

The effects of the plasminogen pathway on scar tissue formation.

OBJECTIVES/HYPOTHESIS: The authors sought to determine the role of the plasminogen pathway in wound healing. They hypothesized that decreased fibrin degradation may lead to increased collagen deposition. Presuming that the degree of histopathological abnormality correlates with the aesthetic appearance of the scar, we conducted a study that attempted to determine the histopathological appearance of scar tissue in mice with and without impaired function of the plasminogen pathway. STUDY DESIGN: Mice with and without deficiencies in the plasminogen pathway underwent surgery. The role of the plasminogen pathway in wound healing was studied by analysis of scar tissue formation using the methods described. METHODS: A 2-cm incision was made on the dorsum of mice with and without specified genetic deficiencies in the plasminogen pathway. After the animals were killed, the tissue was harvested, fixed, and prepared using hematoxylin and eosin as well as trichrome stains. Histopathological analysis and scoring were performed by two separate investigators in a blinded manner. Student's t test was used to determine statistical significance between groups. RESULTS: A statistically significant difference in collagen orientation was noted between mice with impaired plasminogen pathway function and the wild-type (control) group (P =.0163). A statistical trend toward improved wound healing for plasminogen-deficient mice was found for overall histomorphological score (P =.0706). CONCLUSION: The role of the plasminogen pathway in wound healing is one that should be noted and may lead to the development of new therapies that reduce scar tissue formation. Hence, the role of other thrombolytic and anti-thrombolytic agents in wound healing should be further investigated to precisely identify agents that play the most significant role in scar tissue formation.