Papers Form and Function: Homeostatic Regulation of the NMJ. Summary

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1 Papers Form and Function: Homeostatic Regulation of the NMJ Lauren: The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. Lindsay: Ubiquitination-dependent mechanisms regulate synaptic growth and function. Anniek: Highwire restrains synaptic growth by attenuating a MAP kinase signal. Ayumi: Pre- and post-synaptic abnormalities associated with impaired neuromuscular transmission in a group of patients with 'limb-girdle myasthenia' Summary Neuromuscular transmission/quantal Analysis Problem The neuromuscular junction... [is] an experimentally favourable object whose study could throw considerable light on synaptic mechanisms elsewhere - Sir Bernard Katz 3. In an experiment on an isolated flexor digitorum brevis nerve-muscle preparation dissected from a mouse, intracellular microelectrode recordings were made of spontaneous miniature endplate potentials (MEPP). Endplate potentials (EPP) were then evoked by nerve stimulation at a frequency of 1 Hz. In total, 97 of the stimuli applied to the nerve evoked an EPP but 3 stimuli failed to evoke any EPP. The following mean data with their standard deviations were obtained: Mean MEPP amplitude (± SD) : 1.20 ± 0.72 mv Mean EPP amplitude (± SD) : 4.25 ± 2.42 mv a) Speculate on the ratio of Ca 2+ to Mg 2+ ions in the medium bathing this preparation. Intracellular Extracellular Ca nm 1.2 mm Mg mm 1.5 mm Ratio 1.25 x 10^ Neuromuscular transmission/quantal Analysis Problem b) Calculate the mean quantal content of the EPP using the Direct, Variance and Failures Methods. 1. Direct method: Under favourable conditions, both MEPPs and EPPs can be recorded in sufficient numbers to allow cross checking of the quantal content of EPPs, comparing Poisson statistics with direct estimation of the mean quantal content Neuromuscular transmission/quantal Analysis Problem 2. Variance method Another property of the Poisson distribution is that its variance equals its mean. From this it can be derived that: m = (mean EPP amplitude)^2/(variance of EPP amplitudes) m = (mean EPP amplitude) / (mean MEPP amplitude) This method is not always possible for various technical reasons, e.g the MEPPs might be very infrequent; or they may be too small, buried in the noise of the recording system; or the mean quantal content may be large, resulting in nonlinear summation of EPPs (see below). Applying the Poisson equation alone is sometimes sufficient in such cases. There are two methods of estimating quantal content based on the Poisson distribution: the Method of Failures and the Variance Method.

2 Neuromuscular transmission/quantal Analysis Problem 3. Method of Failures If the mean quantal content is low enough (as in the examples above), a significant fraction of stimuli will fail to evoke a response. This represents the P(0) expression in the Poisson distribution: P(0) = exp (-m). m0/0! Neuromuscular transmission/quantal Analysis Problem c) What does the standard deviation of the MEPP amplitude (quantal size) indicate and how might this affect the estimation of mean quantal content? since, by mathematical definition, both m0 and 0! are equal to 1 : P(0) = exp (-m) Taking natural logartithms : Ln (P0) = -m Substituting for P(0)=(Number of Failures)/(Number of Stimuli) and rearranging: m = Ln (Stimuli/Failures) Neuromuscular transmission/quantal Analysis Problem d) Give one other possible reason for a low quantal content, in the contexts of health and disease. Lambert-Eaton Myasthenic Syndrome S.J. Wood and C.R. Slater (1997) The Neuromuscular Junction in Health and Disease Lauren Tryggvason I. Introduction & Aim II. Methods III. Results IV. Conclusions V. Strengths and Weaknesses VI. Future Research Safety factor more quanta are released during neuromuscular transmission than is required for AP generation = functional, reliable, flexible EPP/mEPP transient depol. of postsynaptic membrane (+ve in) EPC current (ion flow) that causes EPP Quantal content # vesicles released in response to stimulus

3 Postsynap3c folds folding of postsynap3c membrane at vertebrate NMJ, contain AChRs and VGSCs (Ruff, 2003) Previous studies: folds create a high-resistance pathway that concentrates current flow on VGSCs = amplifies NT action This study: What is their function? How do they contribute to effective neuromuscular transmission? - Look at the NMJ safety factor - express as quanta released per nerve impulse - compare between fast- and slow-twitch muscle fibres Previously reported safety factor of ~2 Safety factor: fast-twitch>slow-twitch depends on differences on pre- and postsynaptic structures, e.g. postsynaptic folds? If the function of the postsynaptic folds and the VGSCs within them is to enhance the efficacy of neuromuscular transmission then this enhancement may be greater in fast-twitch than in slow-twitch muscles Rat soleus (slow-twitch) and extensor digitorum longus (EDL, fast-twitch) nerve-muscle preparation, incubated in µ- conotoxin or d-tubocurarine Electrophysiological recording: Glass microelectrodes used to make intracellular recordings of - resting potential - two-electrode voltage-clamp (-75mV) measured spontaneous and evoked EPCs 2. Measuring quantal content: A. spontaneous MEPPs, nerve stimulated (suction electrode, 1Hz, 60s) = EPPs B. Voltage-clamp (-75mV) recordings of mepcs and EPCs. Calculate quantal content 3. Measuring threshold: A. nerve-evoked AP threshold: dtc partial transmission block voltageclamp (-75mV) stimulate nerve (0.2 Hz) = threshold EPCs 4. Safety factor calculations: compare quantal content from nerve stimulation to quantal content at threshold (voltage/charge) m v = EPP corr /MEPP B. Injected current pulse at NMJ, recorded peak amplitude just before AP elicited mean EPP amplitude (corrected) mean MEPP amplitude m Q = Q EPC /Q MEPC C. Measure safety factor without postsynaptic specializations: inject current pulse into extrajunctional area, record threshold value before AP initiation mean charge/epc mean charge/quantum

4 5. Structural analysis: Muscle fibre diameter light microscopy of 8µm haematoxylin-stained cryostat sections NMJ area bundles of 5-10 muscle fibres, identify regions of high cholinesterase activity, sum t determine area Postsynaptic folds morphological charactersitics identified using EM Soleus muscle diameters and NMJ areas 50% larger than EDL, but EDL releases >2x NT/area of synap3c contact than soleus No significant differences in postsynap3c fold architecture. So, EDL safety factor affected by increased NT release

5 Postsynaptic folds: - multiply safety factor by two - amplify EPC/aid depolarisation (N/XJ or J) (important for effect neuromuscular transmission (N/XJ)) - fast-twitch have greater safety factors than slow-twitch, but morphologically similar Why is the safety factor so high and does the paper provide sufficient evidence for their explanation? - Previously reported as ~2 - Used more accurate technique, physiologically relevant postsynaptic response to the nerve impulse (not injected currents) - Experimental stimulation frequencies not typical of in vivo activity: 0.2-1Hz vs. 20Hz (soleus) and 60-80Hz (EDL) as frequency increases, quantal content decreases = ration between quantal content and threshold decreases = reduced to 2? STRENGTHS WEAKNESSES Novel approach (EPC vs. EPP and nerve impulse vs. injected current), more direct, more accurate (?) Good checks/sample selection fibres can be damaged by electrodes, so measure resting potential and input resistance to ensure reliability Used two approaches (voltage and charge) to measure same parameter (quantal content) Quantification of morphology Are their results relevant in vivo? Are their results actually more accurate? Leap in logic? Detailed descriptions of measurement/analysis of quantal content etc, less description of how postsynaptic folds may actually account for safety factor title of paper! Figures What happens if postsynaptic folding/safety factor is disrupted? Is safety factor conserved? In vivo recordings? How exactly do postsynaptic folds contribute to the safety factor? Myasthenia gravis alters safety factor: loss of membrane/ach-rs and VGSCs = PPs and hreshold (Ruff, 2008) 5-9x excess in Drosophila (DiAntonio, 2005)? Multifactorial, older studies, effects best seen when comparing fast and slow twitch muscles. DiAntonio, A. & Marrus S.B. Investigating the safety factor at an invertebrate neuromuscular junction. J Neurobiol 2005: 63; MINI-SYMPOSIUM 3: HOMEOSTATIC REGULATION OF STRUCTURE AND FUNCTION AT THE NMJ (By Ayumi Tomari) Ruff, R.L. Neurophysiology of the Neuromuscular Junction: Overview. Ann. NY Acad. Sci 2003: 998; Ruff, R.L. & Lennon, V.A. How myasthenia gravis alters the safety factor for neuromuscular transmission. J Neuroimmunol 2008: ; Slater, C.R. & Wood, S.J. Safety factor at the neuromuscular junction. Progress in Neurobiology 2001: 64; Slater, C.R. & Wood, S.J. The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. J. Physiology 1997: 500;

6 CONTENTS Background Aims of the study Methods Results Treatment Main Findings Strengths and Weaknesses Burning Question Summary Limb-Girdle Myasthenia (LGM) Early onset (during infancy/childhood) Progressive weakness of proximal limb muscles No ocular impairment Characteristic waddling gait Autoimmune and familial aetiologies One study had ~20% LGM patients with increased AChR antibodies Consanguineous parents or affected siblings; genetic origin? Previous study of childhood myasthenia in 1971 (20 year period) Electromyography : Clear evidence of impairment No clear evidence of the cellular, sub cellular or molecular basis of that impairment Aims Identify any structural and functional abnormalities in the NMJ which account for impaired neuromuscular transmission Is the defect pre- or post-synaptic? Methods Patients 8 LGM patients from an ongoing study of neuromuscular diseases Control: Patients with idiopathic muscle pain or adult-onset muscular dystrophy- no obvious neurological involvement Nerve conduction studies Electromyography (Bicept brachii [BB], etensor digitorum communis [EDC], vastus lateralis [VL] and tibialis anterior [TA]) Repetitive nerve stimulation (on proximal and non-proximal muscles) Single-fibre EMG on the EDC and VL during weak voluntary contraction Motor-point biopsy from the top part of VL Muscle fibres and its associated nerves Intracellular recording (EPPs/mEPPs, EPCs/mEPCs) Light microscopy Motor nerve terminals, AChR density, AChE activity Electron microscopy ChE activity Methods (2) Immunocytochemistry Antibody labelling: AChE, s-laminin, alpha/beta AChR subunit, rapsyn, voltage-gated Na channel, ankyring, dystrophin, utrophin, syntrophin, neuregulin and so on. DNA analysis Full screening for mutations in genes (usually involved in congenital myasthenic syndromes, CMS) by bi-directional DNA sequencing for all exons and promoter regions Results: Clinical features Equal numbers of females and males 2 of them with family history but no diagnostic of autosomal inheritance Serum antibodies for AChR were not detected All 8 LGM patients had progressive muscle weaknesses with predominant limb-girdle distribution varied over a period of weeks to months Oculo motor weakness absent; slight ptosis and facial weakness Distinct swaying/ waddling gait

7 Repetitive Nerve Stimulation All LGM patients displayed decrements of compound muscle action potential (CMAP) at 3Hz stimulation. Pronounced decrement in proximal muscles (elbow and shoulder, 41%) than in nonproximal muscles (hands and feet, 19%) Further emphasises the point that neuromuscular transmission in proximal limb muscles are predominantly impaired. Results - electrophysiology Mean EPP amplitude in LGM- 46% of controls Mean mepp amplitude- 68% of controls Mean quantal content- 59% of the control value Significant correlation between EPP amplitude and abnormal muscle fibre pairs Suggests that the reduction in EPP amplitude accounted for observed weakeness of muscle in vivo Results - electrophysiology Significant EPC reduction, but mepc is normal relative to control Possibly due to slightly greater diameter of muscle fibres of LGM patients- hence, decreased input resistance of muscle fibre Results- light microscopy A/B- Motor nerve terminals Smaller and less compact in LGM C/D- AChR density Labelled by fluoroscent rhodamine-alphabungarotoxin No qualitative difference Mean value of fluoroscene label bindnig sites per NMJ- 40% of that in controls E/F- AChE activity ChE area is only 55% of that in control

8 sults- electron microscopy A/B: Control patients C/D: LGM patients Reduction in postsynaptic folds in C/D. Post-synaptic folds & V-gated Na+ channels amplify EPC Folds usually lie outside of the region of close contact with the nerve Variety between patients and also variety within a single patient in terms of folds (F/G) E: Tubular aggregates H/I: Distribution of AChRs- LGM and control. There was no signifiance difference when comparing the average ratios of quantal content/synaptic area for LGM and control samples Quantal release per unit area of synaptic contact is not lower than normal Results - others Protein localisation studies To identify molecular defects underlying structural/functional abnormalities at NMJs of LMG patients. Intensity of immunolabelling INCREASED at the NMJ realtive to non-junctional areas of the muscle (in both LGM and control) DNA analysis Identify abnormalities in genes commonly linked with congenital myasthenic syndromes No mutations were identified Main Findings Combination of clinical features to differentiate from other Congenital Myasthenic Syndromes (CMS) Onset in infancy/childhood, proximal limb muscle weakness and waddling gait Abnormalities in common within the patients: Reduced NMJ sizes Reduced ACh output and post-synaptic folds Increased threshold for muscle fibre action potential generation Other features were normal: Presence of key proteins and genes encoding them Size and kinetics of mepcs Quantal release per unit area of synaptic contact Primary targets = Elements of the mechanisms which determine NMJ size and conformation Both pre- and post-synaptic areas are involved Analysis of the paper Strengths Sufficient amount of qualitative analysis (light microscopy, immunolabeling) Quantitative analysis (Conveyed in the tables) Patients were clinically distinguishable from other CMS patients Weaknesses Small sample size Patients did not represent all of those reported with LGM Some studies suggest autoimmune aetiology (AChR antibody serum was present) Biopsy samples came from different parts (inconsistent observation) BBQ: What is wrong with the synaptic size-strength relationship in CMS and why are only some muscles affected? Synaptic size-strength relationship Nerve-muscle contacts in LGM half of control Reduction in the density of post-synaptic AChR- reduced amplitude of mepp Reduced ACh output and post-synaptic folding- smaller NMJ Selective impairment of limb-girdle muscles is still unknown Possible theories: Unknown autoimmune antibodies or specific immune profiles Future studies: Targeted gene analysis of LGM could identify the aetiology of LGM

9 Ubiquitination-dependent mechanisms regulate synaptic growth and function. A DiAntonio, AP Haghighi, SL Portman, JD Lee, AM Amaranto and CS Goodman Contents Introduction Methods/Results Conclusions Big Burning Question Take Home Points Introduction Ubiquitination Fat facets (faf) Highwire (hiw) Methods A genetic screen was used to obtain two cell lines: EP(3)381 and EP(3)3520. Wild Type This paper aims to show that both positive and negative ubiquitin-dependent mechanisms regulate synaptic development at the Drosophila NMJ. elav-gal4/ EP(3)381 Both of these cell lines overexpress faf leading to synaptic overgrowth in the nervous system. Methods/Results (1) Anatomical Analysis Methods/Results (2) Analysed the spontaneous and evoked neurotransmitter release Although increased NMJ size, there is reduced amplitude of evoked excitatory junctional potentials (EJPs)

10 Methods/Results (2) cont. Small decrease in amplitude of mejps, but large decrease in quantal content. Indicates a presynaptic defect in neurotransmitter release. Methods/Results (3) Overexpression of yeast UBP2 in the Drosophila NMJ results in synaptic overgrowth and reduced presynaptic transmitter release (P= <0.001). Synaptic development is affected by overexpression of deubiquitinating proteases. Methods/Results (3) cont. Methods/Results (4) In a genetic interaction performed 12 lethal enhancers were identified. All 12 were alleles of the highwire (hiw) gene and shared the synaptic overgrowth phenotype. This shows that ubiquitination may have a central role in regulation of synaptic growth and function. Methods/Results (5) Double mutants were generated between loss-of-function alleles of hiw and faf. Two different loss-of-function alleles of faf both suppress the physiological phenotype of hiw, resulting in a more the doubling of quantal content and mejp frequency. Methods/Results (5) cont. faf activity in a hiw background inhibits neurotransmitter release. faf mutants do not suppress hiw synaptic overgrowth The inability to suppress the small quantal size may be a consequence of the synaptic overgrowth seen both in hiw mutants and faf overexpression.

11 Methods/Results (6) Disrupted a glutamate receptor in a hiw mutant. Showed that postsynaptically there was: - an 18% decrease in quantal size - a compensatory 30% increase in quantal content This suggests that homeostatic and ubiquitin-dependent regulation are mechanically distinct. Conclusions Ubiquitin dependent mechanisms regulate synaptic development at the Drosophila NMJ and that both positive and negative regulators of ubiquitin-dependent mechanisms control the synaptic structure and function. This paper proposes that hiw-dependent ubiquitination controls the activity of certain regulatory molecules that can be deubiquitinated by both faf and other deubiquitinating proteases. BBQ- Does a homologous highwire/fat-facets system occur in mammals (including man) and could it be a therapeutic target? Highwire homologues in mammals- Phr and Pam Fat facets homologue in mammals- FAM Does not appear to be a conserved system between the two of them. Pam involved in Tuberous Sclerosis Strengths and Weaknesses Good use of figures and statistical tests Logical progression through experiments, and covered a wide range of tests Overall, good paper A few cases where data was not shown No hypothesis, therefore unable to prove right or wrong Further Studies Find out more about the mammalian homologues of highwire (Phr and Pam) and fat-facets (FAM). Look into therapeutic targets in the regulation of presynaptic development. Take home points The loss-of-function effect of highwire has the same phenotype as the fat facets gain-of-function mutants. This suggests that the balance between ubiquitination and deubiquitination is important for regulating synaptic growth and development.

12 Highwire Restrains Synaptic Growth by Attenuating a MAP Kinase Signal Catherine A. Collins, Yogesh P. Wairkar, Sylvia L. Johnson, and Aaron DiAntonio, Neuron vol 51, page 57-69, 2006 Background Highwire Presynaptic regulator of synapse growth Down regulates signaling proteins that promote growth Mutations/knock-out cause synaptic overgrowth at NMJ Homologues: C.elegans (rpm-1), Zebrafish (esrom), mouse (phr1) Kda E3 ubiquitin ligase activity Wallenda MAPKKK Wallenda protein acts as a down stream target of Highwire JNK, Fos act down stream of Wallenda Highwire = Wallenda Synaptic growth Aim To identify the downstream signaling pathway of Highwire for the regulation of synaptic growth. Hypothesis Highwire restrains synaptic growth by down regulating levels of a synaptic protein that promotes synaptic growth. A Genetic Screen for Mutations that Suppress the highwire Synaptic Overgrowth Phenotype To identify genes whose dosage is important for synaptic overgrowth Genetic screen for mutations that can dominantly suppress the highwire phenotype Highwire (loss of function) + faf = Lethal GAL4- UAS Downstream targets should rescue lethality 2 suppressor mutations on single locus on chromosome 3 Loss of function alleles Figure 1 Wallenda

13 Wallenda mutations completely suppress the highwire synaptic overgrowth phenotype Glutamatergic type 1 NMJ DVGLUT synaptic vesicle marker Wallenda mutations Parameters supressed Remove 1 allele, suppresed by 50% Remove both alleles suppressed by 100% Highwire, wallenda double mutant Similar to WT Wallenda gene for Highwire overgrowth Comparison to Suppression by wit Mutants BMP/TGF-β signalling pathway stops highwire overgrowth- related to wallenda? Mutations in Wit (type 2 receptor in TGF pathway) Reduction in synaptic growth Do not reduce bouton or vesicle size Wallenda and TGF suppress highwire through different mechanisms wallenda Suppresses Synaptic Overgrowth Caused by Overexpression of fat facets Both Gain of function faf and loss of function highwire influence pathway. To see if wallenda can suppress faf induced overgrowth. Wallenda is needed for overgrowth Figure 2 Wallenda Does Not Suppress the highwire Defect in Synaptic Function Can Wallenda mutants suppress defects of Highwire? Wallenda mutants have normal synaptic function Wallenda mutants supress Highwire quantal size defect Wallenda mutants do not supress Highwire vesicle number defect Highwire regulates synaptic physiology through a different pathway not requiring wallenda. Figure 3

14 Wallenda is a Conserved MAPKKK Wallenda is mapped on 3rd chromosome where 4 genes are located Wnd1- kinase domain Wnd2- stop codon Wnd3 - suppressor mutation (loss of function) that can suppress highwire overgrowth. Homolog DLK, LZK Figure 4 Endogenous Wallenda Protein Localizes to Synapses and Is Regulated by Highwire In situ hybridization first detect wallenda at stage 13 in embryonic CNS To determine where wallenda localizes Αlpha wallenda stain Stains neuropil Wnd3 mutants showed no neuropil staining No difference in WT and highwire staining Loss of highwire changes staining in nerve chord WT wallenda not detected in neuropil Highwire mutant increased neuropil staining Highwire contols wallenda in larval stage when NMJ grows a lot Wallenda is regulated by ubiquitination Highwire affects levels of wallenda protein in NMJ Highwire Can also Influence the Levels of Transgenic Wallenda Protein Highwire and faf affect wallenda stain directly due to regulation of protein or indirectly by regulating wallenda gene expression Highwire mutant with wallenda is lethal Highwire normally counteracts wallenda Transgene made to overexpress without causing death Does removal of highwire affect level of transgene? X6 increase in wallenda at NMJ Highwire down regulates Wallenda protein Figure 5 Wallenda is Sufficient to Confer Synaptic Overgrowth To test if overexpression of wallenda causes overgrowth Neuronal overexpression of wallenda is enough to induce synaptic overgrowth

15 Figure 6 Synaptic Overgrowth Requires JNK Signaling To see if p38 or Bsk are required for highwire overgrowth Wallenda gain of function is suppressed by Bsk Overgrowth in highwire mutant requires JNK signaling Figure 7 Synaptic Overgrowth Requires the Fos Transcription Factor AP-1 complex of Fos and Jun TF Down stream affecter of JNK mediated changes in gene expression To see if for or Jun are required for highwire dependent synaptic overgrowth Fos acts separately from Jun Requirement of Fos for overgrowth means highwire involves changes in gene expression rather than local changes in synaps Conclusion Wallenda (MAPKKK) levels are controlled by Highwire and activity of ubiquitin hydrolases Wallenda is needed for highwire dependent synaptic overgrowth JNK and Fos are downstream targets are also required for overgrowth In absence of Highwire pathway is over active and leads to changes in gene expression resulting in excessive synaptic growth