The mitochondrial protein Sideroflexin 3 (SFXN3) influences neurodegeneration pathways in vivo

Synapses are a primary pathological target in neurodegenerative diseases. Identifying therapeutic targets at the synapse could delay progression of numerous conditions. The mitochondrial protein SFXN3 is a neuronally-enriched protein expressed in synaptic terminals and regulated by key synaptic proteins, including α-synuclein. We first show that SFXN3 uses the carrier import pathway to insert into the inner mitochondrial membrane. Using high-resolution proteomics on Sfxn3-KO mice synapses, we then demonstrate that SFXN3 influences proteins and pathways associated with neurodegeneration and cell death (including CSPα and Caspase-3), as well as neurological conditions (including Parkinson’s disease and Alzheimer’s disease). Over-expression of SFXN3 orthologues in Drosophila models of Parkinson’s Disease significantly reduced dopaminergic neuron loss. In contrast, the loss of SFXN3 was insufficient to trigger neurodegeneration in mice, indicating an anti- rather than pro-neurodegeneration role for SFXN3. Taken together, these results suggest a potential role for SFXN3 in the regulation of neurodegeneration pathways

Bioenergetic status modulates motor neuron vulnerability and pathogenesis in a zebrafish model of spinal muscular atrophy

Degeneration and loss of lower motor neurons is the major pathological hallmark of spinal muscular atrophy (SMA), resulting from low levels of ubiquitously-expressed survival motor neuron (SMN) protein. One remarkable, yet unresolved, feature of SMA is that not all motor neurons are equally affected, with some populations displaying a robust resistance to the disease. Here, we demonstrate that selective vulnerability of distinct motor neuron pools arises from fundamental modifications to their basal molecular profiles. Comparative gene expression profiling of motor neurons innervating the extensor digitorum longus (disease-resistant), gastrocnemius (intermediate vulnerability), and tibialis anterior (vulnerable) muscles in mice revealed that disease susceptibility correlates strongly with a modified bioenergetic profile. Targeting of identified bioenergetic pathways by enhancing mitochondrial biogenesis rescued motor axon defects in SMA zebrafish. Moreover, targeting of a single bioenergetic protein, phosphoglycerate kinase 1 (Pgk1), was found to modulate motor neuron vulnerability in vivo. Knockdown of pgk1 alone was sufficient to partially mimic the SMA phenotype in wild-type zebrafish. Conversely, Pgk1 overexpression, or treatment with terazosin (an FDA-approved small molecule that binds and activates Pgk1), rescued motor axon phenotypes in SMA zebrafish. We conclude that global bioenergetics pathways can be therapeutically manipulated to ameliorate SMA motor neuron phenotypes in vivo

Microarray analysis of differentially vulnerable motor neuron pools reveals fundamental differences in their basal molecular composition.

<p>(A) Schematic illustration of experimental design (TA, tibialis anterior; EDL, extensor digitorum longus; GS, gastrocnemius; Vul, vulnerable MNs, Res, resistant MNs; Int, intermediate phenotype MNs). (B) Volcano plots of differentially expressed transcripts in resistant compared to vulnerable MN pools, intermediate compared to vulnerable MN pools, and resistant compared to intermediate MN pools. (C) Ratio trending analysis: transcripts that were significantly changed (p<0.05) between resistant (EDL) and vulnerable (TA) groups with a differential trending value in the intermediate (GS) group were first identified after which the data set underwent enrichment analysis to reveal enriched biological pathways. Graph shows an example of genes in one enriched biological pathway (mitochondrial electron transport chain genes). Note that transcripts showed highest expression levels in resistant (EDL) neurons, with a decreasing level of expression as the vulnerability status of the groups increased (GS through to TA). (D) qPCR validation for 3 distinct mitochondrial genes confirming up-regulation in disease-resistant MN pools (N = 3), Unpaired two tailed student <i>t-test</i> (* P<0.05). (E) Bar chart (mean & s.e.m.) showing a reduction in ATP in the spinal cord of early and late-symptomatic SMA mice compared to littermate controls using an ATP assay (N = 3 spinal cords per genotype).</p

Pgk1 expression is pathologically relevant in mouse and zebrafish models of SMA.

<p>(A) Expression of PGK1 protein in the spinal cord, skeletal muscle, sciatic nerve and heart of late-symptomatic P8 SMA mice. Protein levels were quantified and normalized to an appropriate loading control. (B) Bar chart (mean & s.e.m.) showing a significant reduction in PGK1 protein levels in SMA mouse spinal cord and sciatic nerve. N = 6 SPC per genotype. N = 3 muscle per genotype. N = 7 sciatic nerves per genotype. N = 3 hearts per genotype (C) Knockdown of Pgk1 in zebrafish induced an axonal outgrowth phenotype (middle panel arrow) similar to smn knockdown (arrow bottom panel) and also produced swellings in the tips of outgrowing axons indicative of axonal transport deficiencies. Scale bars = 50 μM (D) Quantification of axonal outgrowths showed a significant increase in truncated motor axons in pgk1 and smn morphants compared to controls. (E) Efficiency of <i>pgk1</i> knockdown in embryos was shown by western blot embryos normalized to an appropriate loading control (N = 3 per group, batches of 30 pooled zebrafish embryos per lane). N = 20 embryos per group. Unpaired two-tailed students <i>t-test</i> * p<0.05, ** p<0.01 *** p<0.001 **** p<0.0001.</p

Primer sequences used in quantitative PCR.

<p>Primer sequences used in quantitative PCR.</p

Enrichment analysis of microarray data reveals biological pathways changed between disease-resistant and vulnerable motor neuron pools.

<p>Enrichment analysis of microarray data reveals biological pathways changed between disease-resistant and vulnerable motor neuron pools.</p

Overexpression or pharmacological activation of pgk1 rescues motor neuron phenotypes in smn morphant zebrafish.

<p>(A) Representative confocal micrographs of primary motor neuron axons exiting the spinal cord in control (top), <i>smn</i> morphant (middle) and <i>smn</i> morphant over-expressing <i>pgk1</i> (bottom) Tg(<i>hb9</i>:GFP) zebrafish embryos. Note the presence of an axonal outgrowth/branching phenotype associated with <i>smn</i> knockdown (arrow heads) that is reduced in the <i>pgk1</i> over-expressing animals. Scale bars = 50 μM. (B) Overexpression of Pgk1 in <i>smn</i> morphant zebrafish at 30 hpf led to a significant increase in normal motor axons and significant decrease in severe axonal outgrowth phenotypes compared to single <i>smn</i> MO injected embryos. (C) Representative confocal micrographs of motor neuron axons exiting the spinal cord in control (top), <i>smn</i> morphant (middle) and <i>smn</i> morphant treated with 2.5 μM terazosin (bottom) Tg(<i>hb9</i>:GFP) zebrafish embryos. Note how the presence of the axonal outgrowth/branching phenotype associated with <i>smn</i> knockdown (arrow heads) is reduced in the terazosin-treated animals. (D) Bar chart (mean & s.e.m) showing activation of Pgk1 by treatment with 2.5 μM terazosin in <i>smn</i> morphant zebrafish at 30 hpf led to a significant increase in normal motor axons and significant decrease in severe axonal outgrowth phenotypes compared to untreated <i>smn</i> MO injected embryos. Unpaired two-tailed student <i>t-tests</i> * p<0.05, ** p<0.01 *** p<0.001. n = 20 embryos per group.</p

Clinical autonomic nervous system laboratories in Europe: A joint survey of the European Academy of Neurology and the European Federation of Autonomic Societies

Background and purpose: Disorders of the autonomic nervous system (ANS) are common conditions, but it is unclear whether access to ANS healthcare provision is homogeneous across European countries. The aim of this study was to identify neurology-driven or interdisciplinary clinical ANS laboratories in Europe, describe their characteristics and explore regional differences. Methods: We contacted the European national ANS and neurological societies, as well as members of our professional network, to identify clinical ANS laboratories in each country and invite them to answer a web-based survey. Results: We identified 84 laboratories in 22 countries and 46 (55%) answered the survey. All laboratories perform cardiovascular autonomic function tests, and 83% also perform sweat tests. Testing for catecholamines and autoantibodies are performed in 63% and 56% of laboratories, and epidermal nerve fiber density analysis in 63%. Each laboratory is staffed by a median of two consultants, one resident, one technician and one nurse. The median (interquartile range [IQR]) number of head-up tilt tests/laboratory/year is 105 (49–251). Reflex syncope and neurogenic orthostatic hypotension are the most frequently diagnosed cardiovascular ANS disorders. Thirty-five centers (76%) have an ANS outpatient clinic, with a median (IQR) of 200 (100–360) outpatient visits/year; 42 centers (91%) also offer inpatient care (median 20 [IQR 4–110] inpatient stays/year). Forty-one laboratories (89%) are involved in research activities. We observed a significant difference in the geographical distribution of ANS services among European regions: 11 out of 12 countries from North/West Europe have at least one ANS laboratory versus 11 out of 21 from South/East/Greater Europe (p = 0.021). Conclusions: This survey highlights disparities in the availability of healthcare services for people with ANS disorders across European countries, stressing the need for improved access to specialized care in South, East and Greater Europe