Retrograde mitochondrial transport regulates mitochondrial biogenesis in zebrafish neurons

Retrograde mitochondrial transport is required for mitochondrial biogenesis in zebrafish neurons | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Retrograde mitochondrial transport is required for mitochondrial biogenesis in zebrafish neurons Angelica E Lang , Chris Stein , Roger Schultz , View ORCID Profile Catherine M Drerup doi: https://doi.org/10.1101/2025.09.29.679307 Angelica E Lang 1 Department of Integrative Biology; University of Wisconsin-Madison; Madison , WI 53706 2 Genetics training program; University of Wisconsin-Madison; Madison , WI 53706 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Chris Stein 1 Department of Integrative Biology; University of Wisconsin-Madison; Madison , WI 53706 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Roger Schultz 1 Department of Integrative Biology; University of Wisconsin-Madison; Madison , WI 53706 3 Neuroscience training program; University of Wisconsin-Madison; Madison , WI 53706 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Catherine M Drerup 1 Department of Integrative Biology; University of Wisconsin-Madison; Madison , WI 53706 2 Genetics training program; University of Wisconsin-Madison; Madison , WI 53706 3 Neuroscience training program; University of Wisconsin-Madison; Madison , WI 53706 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Catherine M Drerup For correspondence: drerup{at}wisc.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract To maintain a functional mitochondrial population in a long-lived cell like a neuron, mitochondria must be continuously replenished through the process of mitochondrial biogenesis. Because most mitochondrial proteins are nuclear encoded, mitochondrial biogenesis requires communication between mitochondria and the nucleus. This can be a challenge in a large, compartmentalized cell like a neuron in which a large portion of the mitochondrial population is in neuronal compartments far from the nucleus. Using in vivo assessments of mitochondrial biogenesis in zebrafish neurons, we determined that mitochondrial transport between distal axonal compartments and the cell body is required for sustained mitochondrial biogenesis. Estrogen-related receptor transcriptional activation links transport with nuclear expression of mitochondrial genes. Together, our data support a role for retrograde feedback between axonal mitochondria and the nucleus for regulation of mitochondrial biogenesis in neurons. Introduction Mitochondria are critical for neuronal function and survival. These organelles have many essential roles, including production of ATP, calcium buffering, and the production of metabolites that can function as signaling molecules 1 . For a neuron to maintain a functional mitochondrial population, mitochondria must be replenished through the process of mitochondrial biogenesis 2 . In other cell types, such as myocytes and adipocytes, indicators of mitochondrial health and function like AMP/ATP and NAD+/NADH ratios as well as calcium signaling stimulate the transcriptional control of mitochondrial biogenesis in the nucleus 3 – 6 . These upstream signals can activate transcriptional regulators like the PGC-1 (Peroxisome Proliferator-activated Receptor Gamma Coactivator 1) family of transcriptional coactivators 7 . PGC-1α, considered the master regulator of mitochondrial biogenesis, can be activated by phosphorylation or de-acetylation by AMP-activated protein kinase (AMPK) or Sirtuin 1 (SIRT1) respectively 3 , 4 , 6 . AMPK activity is stimulated by increases in the AMP/ATP ratio while SIRT1 activity depends on local NAD+ levels. Once activated, PGC-1α can induce the expression of nuclear-encoded mitochondrial proteins through the activation of transcription factors like the nuclear respiratory factors (NRF1/2) or the estrogen related receptors (ERRα/β/γ) 7 – 10 . How neurons, with mitochondrial populations spread throughout dendritic and axonal arbors, oftentimes large distances from the nucleus, sense mitochondrial health and function to activate these transcriptional pathways is an open and critical question 2 . Mitochondrial biogenesis requires the division and expansion of existing organelles to increase mitochondrial mass. This requires the coordination of several cellular processes. Mitochondrial DNA (mtDNA) must be replicated and the mtDNA nucleoids segregated, after which mitochondrial fission occurs at or near the middle of the organelle 11 , 12 . Daughter mitochondria are expanded through transcription, translation, and mitochondrial import of nuclear encoded gene products as well as cellular and mitochondrial derived lipids 2 , 13 . Mitochondria also synthesize proteins encoded by mtDNA (thirteen in vertebrates) in the mitochondrial matrix. Given the dependence on coordinated mitochondrial and nuclear gene expression, it is unsurprising that a substantial amount of mitochondrial biogenesis occurs in the perinuclear region in non-neuronal cells 14 , 15 . In neurons, evidence of mitochondrial biogenesis, including mtDNA replication and synthesis of mitochondrial proteins, has been observed in both the neuronal cell body and in axons of chick, rat, and drosophila 13 , 16 – 19 . Local mitochondrial biogenesis is thought to support short-term, local changes in energy need at the synapse 16 , 19 , 20 . The respective contributions of cell body-based verses local mitochondrial biogenesis for support of synaptic mitochondrial populations is still a subject of investigation. For both, however, nuclear expression of mitochondrial genes would be required to produce the >1000 proteins necessary for functional organelles. Mitochondria are actively transported between the cell body and axon terminal by microtubule-based transport. In neuronal axons, kinesin motor proteins are responsible for axon terminal directed (anterograde) mitochondrial transport while the Cytoplasmic dynein motor transports mitochondria from the periphery back to the cell body (retrograde) 21 – 24 . A complement of proteins including Trak1/2 and Miro1/2 have been shown to scaffold mitochondria to these motors for transport 25 – 31 . Disrupting mitochondrial transport into the axon impairs axon development and regeneration after injury 32 – 34 . Inhibition of retrograde mitochondrial transport from neurites back to the cell body causes mitochondrial accumulation in axonal and dendritic processes and impaired mitochondrial health at the synapse 35 – 37 . However, the role of retrograde mitochondrial transport in mitochondrial population maintenance in neurons remained undefined. We hypothesized that transport of mitochondria from distal regions of the neuron to the cell body could link mitochondrial population surveillance and regulation of mitochondrial biogenesis frequency in neurons. Using zebrafish neurons, we show that reducing mitochondrial retrograde transport from the axon terminal back to the cell body reduces markers of mitochondrial biogenesis, including mtDNA replication, nuclear transcription of mitochondrial genes, and the production of mitochondrial biomass in the cell body. mtDNA replication is also reduced in the axon terminal, suggesting both local and cell body-based mitochondrial biogenesis may be disrupted. Analysis of mitochondrial turnover at the synapse revealed that reduced mitochondrial biogenesis correlates with reductions in cell body-derived mitochondrial material transported to the axon terminal. Widespread disruption to mitochondrial gene expression in neurons lacking mitochondrial retrograde transport suggests transport plays an important role in regulation of transcriptional programs necessary for mitochondrial biogenesis. We present evidence that this crosstalk is mediated by NAD+/SIRT1-mediated deacetylation of Estrogen related receptors (ERR) which regulates ERR-dependent transcriptional activity. Together, our work supports a model in which the retrograde transport of mitochondria is a powerful signaling mechanism to regulate mitochondrial biogenesis in neurons. Results Inhibiting mitochondrial retrograde transport disrupts markers of mitochondrial biogenesis in neuronal cell bodies We used larval zebrafish posterior lateral line (pLL) sensory neurons to study the dynamics of mitochondrial biogenesis in vivo. The pLL system is composed of afferent neurons and sensory organs called neuromasts situated along the trunk 38 . The cell bodies of pLL neurons are clustered in a ganglion (pLLg) near the head while the afferent axons extend down the body and branch into axon terminals that innervate neuromasts ( Fig. 1a ). Axon extension and synapse formation occurs by 4 days post fertilization (dpf; 39 ), allowing us to use this system to study mitochondrial populations in a fully formed neuronal circuit in the translucent zebrafish larva. To disrupt retrograde mitochondrial transport in pLL neurons, we used an existing mutant line with a unique loss of function mutation in the gene encoding Actr10 ( actr10 nl 15 ; hereafter referred to as actr10; 35 , 37 ). Actr10 is a component of the Cytoplasmic dynein accessory complex dynactin. Complete loss of Actr10 function results in failed cell division, likely due to loss of all dynein motor function 40 . In contrast, the actr10 nl 15 mutation in zebrafish causes loss of mitochondrial interaction with the dynein motor, leading to loss of retrograde (cell body directed) mitochondrial transport and accumulation of mitochondria in axon terminals ( 35 ; Fig. 1b, c ). This disruption is specific to mitochondria, as other retrogradely moving cargos, including late endosomes, lysosomes, and signaling molecules, are transported normally. Additionally, transmission electron microscopy (TEM) analysis showed accumulations of only mitochondria in axon terminals 35 . Intriguingly, in addition to mitochondrial accumulation in the distal axons, we found a severe reduction in mitochondrial density (mitochondrial area/cytosolic area) in the cell body of actr10 neurons ( Fig. 1b, c ). We asked if this loss of mitochondrial density could be due to loss of mitochondrial biogenesis in actr10 mutants. Download figure Open in new tab Figure 1: Mitochondrial cell body density and biogenesis markers are reduced in actr10 mutants. (a) 4 dpf zebrafish transgenic larva carrying the TgBAC(neurod:egfp) nl 1 transgene which labels neurons with GFP. The posterior lateral line gangion (pLLg) and a pLL axon terminal are boxed and magnified. (b) 4 dpf pLLg cell body and pLL axon terminal expressing cytosolic GFP and mitochondria-localized TagRFP (MitoTagRFP, magenta) in wild type and actr10 mutants. (c) Quantification of mitochondrial density (mitochondrial area/cytosolic area; ANOVAs). (d) HCR RNA FISH labeling of pgc1a and tfam mRNA in the pLLg of wild type and actr10 mutants carrying the TgBAC(neurod:egfp) nl 1 transgene (white outline). (e) pLLg mean fluorescence intensity of pgc1a and tfam normalized to background (ANOVAs) in wild type, actr10 , nudc , p150 , and wild type/ actr10 larvae expressing Actr10 in neurons (+ rescue). (f) pLL cell body (outlined) expressing POLG2-GFP and mitoTagRFP (Mito, Magenta). (h) SSBP1 immunostaining in pLL cell body mitochondria, visualized with mitochondria-localized GFP (Mito, magenta; cell outlined). (g, i) Number of POLG2 or SSBP1 puncta normalized to mitochondrial volume (Wilcoxon). Scale bars: (a) full larva = 200 μm, insets = 10 μm; (b, f, & h) = 5 μm; (d) = 10 μm. All data are mean ± SEM and points represent individual larvae. Mitochondrial biogenesis requires coordinated transcription of nuclear encoded mitochondrial genes. This is largely controlled by the PGC-1 family of transcriptional coactivators and their associated transcription factors, including NRF-1/2. Downstream of this transcriptional activation is a plethora of nuclear-encoded mitochondrial genes, including tfam 7 , 10 . We first assessed the expression of pgc1a , pgc1b , nrf1 , and tfam using colorimetric in situ hybridization and found that all were noticeably reduced in actr10 mutants (Supplementary Fig. 1a, b). To quantify gene expression of biogenesis markers in the pLLg, we turned to hybridization chain reaction RNA fluorescence in situ hybridization (HCR RNA FISH; 41 ). We assessed pgc1a and tfam expression and confirmed both were significantly reduced in actr10 mutants ( Fig. 1d, e ). Neuronal rescue of Actr10 in a stable transgenic line ( Tg(5kbneurod:mRFP-actr10) nl 22 ; 35 ) rescued neuronal mitochondrial density (Supplementary Fig. 1c, d) as well as pgc1a and tfam expression ( Fig. 1e ) confirming loss of Actr10 in neurons underlies this phenotype. To determine if the changes to gene expression in actr10 mutants could be explained by general disruptions to retrograde cargo transport, we quantified gene expression in control mutant strains with loss of function mutations in p150a/b (duplicated gene in teleost fish requiring double knockout) and nudc 35 , 42 . p150 is a component of the dynactin complex that, when mutated, disrupts all retrograde cargo transport, including mitochondria 35 , 43 – 45 . NudC is a dynein regulator that, when mutated in zebrafish neurons, disrupts the retrograde transport of late endosomes and autophagosomes without altering the localization or transport of mitochondria 42 . pgc1a and tfam expression were reduced in p150 mutants but unaffected in nudc mutants, further suggesting the decreased expression of these mitochondrial biogenesis markers was due to the loss of retrograde mitochondrial transport ( Fig. 1e ). We next analyzed a critical step during biogenesis, mtDNA replication, using POLG2-GFP, a fluorescently tagged version of the processivity subunit of the mitochondrial DNA polymerase. POLG2-GFP has been previously shown to specifically localize to mtDNA nucleoids actively undergoing replication 12 , 46 . We expressed POLG2-GFP in pLL neurons and quantified the number of puncta, indicative of replicating mtDNA nucleoids, relative to mitochondrial volume. The number of POLG2 puncta was significantly reduced in actr10 pLL cell body mitochondria, indicative of reduced mtDNA replication ( Fig. 1f, g ). To confirm these results, we performed immunolabeling for single stranded binding protein 1 (SSBP1), which transiently associates with mtDNA to facilitate replication 47 . The number of SSBP1 puncta relative to mitochondrial volume was significantly reduced in actr10 pLL cell bodies ( Fig. 1h, i ). This phenotype is not due to loss of mtDNA as the density of nucleoids labelled with the mtDNA nucleoid protein TFAM (transcription factor A, mitochondria; 48 ) is not decreased in actr10 cell body mitochondria (Supplementary Fig.1e, f). As expected, due to the reduced mitochondrial density in the cell body and in line with loss of tfam mRNA based on HCR RNA FISH quantification, overall number of TFAM puncta are reduced in actr10 mutant cell bodies (Supplementary Fig. 1g). mtDNA nucleoid size was unchanged between wild type and actr10 mutants (Supplementary Fig. 1h). Together with our previous results demonstrating no deficits in measures of mitochondrial health and function in actr10 mutant pLL neuronal cell bodies 37 , this suggests decreases in mtDNA replication are not due to mitochondrial stress in the cell body. Mitochondrial biogenesis has also been demonstrated to occur in the distal axon through assessment of mtDNA replication 16 , 19 , 20 . To determine if local mitochondrial biogenesis was also affected in actr10 mutants, we quantified mtDNA replication in the pLL axon terminal. Both POLG2 and SSBP1 puncta were significantly reduced in actr10 mutants, suggesting both cell body and axon terminal mitochondrial biogenesis are disrupted by loss of retrograde mitochondrial transport (Supplementary Fig. 1i-l). Nuclear transcription of mitochondrial genes is disrupted in actr10 mutant neurons Given the changes to mtDNA replication and transcription of mitochondrial biogenesis related genes, we decided to look more broadly at how nuclear gene expression of mitochondrial proteins may be altered in actr10 mutants. We isolated pLL neurons using fluorescence activated cell sorting (FACS) from a novel transgenic line ( Tg(hsp70:eGFP-SILL ) uwd12Tg ; Supplementary Fig. 2a, b) which expresses GFP in pLL neurons. FACS gating parameters were optimized to sort true GFP-expressing cells and exclude autofluorescence particularly apparent in the yolk (Supplementary Fig. c). We then performed bulk RNA-sequencing on FACS sorted cells to analyze the transcriptomes of wild type and actr10 mutant neurons ( Fig. 2a ). Differential gene expression analysis and cross comparisons to the MitoCarta database confirmed that mitochondrial genes are significantly downregulated in actr10 mutants compared to wild type siblings ( Fig. 2b, c ; 49 ). In contrast, there were only 3 mitochondrial genes significantly upregulated (p=0.999, Fisher’s exact test). Gene Set Enrichment Analysis (GSEA) of Gene Ontology terms identified Mitochondrial Protein Complexes to be the most significantly de-enriched term in the dataset ( Fig. 2d and Supplementary Fig. 2d) and KEGG pathway enrichment analysis showed strong enrichment for mitochondrial pathways in downregulated genes (Supplementary Fig. 2e). To confirm our RNA-seq results, we used HCR RNA FISH to quantify pLL ganglion expression of ten downregulated genes and two genes that were not altered in our dataset. These analyses aligned with our RNA-seq data, confirming loss of these mitochondrial transcripts in actr10 mutants ( Fig. 2e-h ). Additionally, we confirmed these results using colorimetric in situ hybridization of a subset of these genes, showing reduced expression of atp5md and cox5ab but not the controls polb and tomm40 (Supplementary Fig. 2f, g). Finally, to assess the specificity of the transcriptomic changes, we included positive and negative control mutant strains p150 and nudc described above 35 , 42 . As predicted, p150 but not nudc mutants have a loss of cox5ab expression ( Fig. 2e, f ). Together, this transcriptomic analysis suggests that mitochondrial retrograde transport modulates nuclear expression of a subset of mitochondrial genes. Download figure Open in new tab Figure 2: Mitochondrial gene expression is downregulated in actr10 mutants. (a) RNA-sequencing strategy. (b) Volcano plot showing differential gene expression between wild type and actr10 mutants. The plot shows -log10(adjusted p-value) vs. log2(Fold Change). Blue dots represent genes significantly downregulated while red dots represent genes significantly upregulated in actr10 mutants. Genes selected for secondary analysis using HCR RNA FISH are labeled. (c) Overlap between downregulated genes and the MitoCarta3.0 database of the mitochondrial proteome (Fisher’s exact test). Human homologs of downregulated zebrafish genes were used for analysis. Duplicated zebrafish paralogues were only counted once. (d) GSEA plot for Mitochondrial Protein Complexes, the most significantly de-enriched gene ontology term in actr10 mutants relative to wild type. (e) Representative images of HCR RNA FISH labeling of cox5ab mRNA in the pLLg (outlined in white) for wild type, actr10 , nudc , and p150 mutants. Scale bar = 10 μm. (f) pLLg mean fluorescence intensity of cox5ab normalized to background (ANOVAs). (g) pLLg mean fluorescence intensity normalized to background for downregulated mitochondrial genes in wild type vs. actr10 mutants (ANOVAs). (h) pLLg mean fluorescence intensity normalized to background for control genes in wild type vs. actr10 mutants (ANOVAs). (f-h) Data plot lines represent the median and quartiles; data points represent individual larvae. Impaired mitochondrial biogenesis decreases the contribution of cell body-derived mitochondria to the axon terminal We next wanted to determine if these changes in measures of mitochondrial biogenesis translated into a decrease in the production of new organelles. In neurons, we predicted that at least a subset of mitochondria produced by biogenesis in the cell body would be transported in the anterograde direction to support the population of mitochondria in the axon terminal 2 . To test this, we used photo-labeling strategies to assess the rate of cell body-derived mitochondrial addition to the axon terminal in wild type animals. We expressed photoactivatable GFP (PA-GFP) localized to the mitochondrial matrix in pLL neurons and locally activated the GFP only in the cell body. Then, we quantified the addition of cell body derived mitochondrial material to the axon terminal at multiple time-points after photo-activation. We found that by 4 to 8 hours post-activation (hpa), ∼50% of the mitochondrial density in the axon terminal contained material that was cell body-derived (Supplementary Fig. 3a-d). Next, we asked if the amount of cell body-derived mitochondrial material transported to the axon terminal correlated with turnover of the axon terminal mitochondrial population. Published data has shown that axon terminal mitochondria are consistently lost over a period of hours in pLL axon terminals 37 . Using precise activation of matrix-localized PA-GFP in axon terminal mitochondria followed by post-activation imaging, we determined that ∼50% of the labeled mitochondrial density is absent from the axon terminal by 8 hpa (Supplementary Fig. 3e-g). This complements the estimated rate of mitochondrial entrance into the axon terminal from the cell body and suggests that cell body-derived mitochondria may play a significant role in maintenance of the axon terminal mitochondrial population. Given the role for cell body-derived mitochondria in axon terminal turnover, we predicted that decreased biogenesis in the cell body would result in a progressive loss of mitochondrial addition to the axon terminal. To test this, we assessed changes to mitochondrial biogenesis and transport in actr10 mutants across multiple timepoints post-axon extension (2, 3, and 4 dpf). Initial axon extension of primary pLL axons is complete by 2 dpf with functional synapses apparent by 4 dpf 38 , 39 , 50 . Assessment of mitochondrial density in axon terminals and cell bodies of actr10 mutants demonstrated that mitochondrial density was elevated in axon terminals as early as 2 dpf (Supplementary Fig. 4a, b) and cell body mitochondrial density was notably depleted by 3 dpf (Supplementary Fig. 4c, d). Quantification of the biogenesis markers pgc1a and tfam using HCR RNA FISH demonstrated that loss of cell body mitochondrial density at 3 dpf corresponded with loss of pgc1a expression, followed by loss of tfam expression by 4 dpf ( Fig. 1d, e and Supplementary Fig. 4e, f). This time course confirms that loss of cell body mitochondrial density correlates with decreased markers of mitochondrial biogenesis. We predicted that the progressive loss of cell body mitochondrial biogenesis in actr10 mutants would result in reduced anterograde mitochondrial transport. Live imaging of mitochondrial movement in pLL axons demonstrated that retrograde transport was disrupted at 3 dpf while anterograde transport frequency was not altered until 4 dpf (Supplementary Fig. 4g, h and Supplementary Movies 1-4). This time series demonstrated retrograde mitochondrial transport is lost first in actr10 mutants, leading to mitochondrial accumulation in the axon terminal. Only after retrograde mitochondrial transport is inhibited does mitochondrial biogenesis fail, leading to loss of cell body mitochondria and reduced anterograde transport of mitochondria towards axon terminals (summarized in Supplementary Fig. 4i). Together, our data suggest that anterograde mitochondrial transport supplies the axon terminal with cell body-derived mitochondria produced through biogenesis. To test this, we directly stimulated mitochondrial biogenesis and analyzed axon terminal mitochondrial addition. To increase mitochondrial biogenesis, we used a stable transgenic zebrafish line overexpressing human PGC-1α in neurons ( Tg(-5kbneurod1:PGC1α-2A-mRFP) uwd9Tg ; 51 ). PGC-1α overexpression in this line increased tfam expression in pLL neurons and has been previously shown to increase mitochondrial density in the axon terminal, confirming increased mitochondrial biogenesis (Supplementary Fig. 4j; 51 ). To determine if increased mitochondrial biogenesis increases the addition of cell body-derived mitochondria to the axon terminal, we used photoconversion and mitochondrial tracking. For this, we used a dual transgenic line expressing PGC-1α ( Tg(-5kbneurod1:PGC1α-2A-mRFP) uwd9Tg ) and mEos, a photoconvertible protein, localized to the mitochondrial matrix ( Tg(-5kbneurod1:mito-mEos) y 586 Tg ; 37 , 52 ). We photoconverted the mitochondrial population in the pLL ganglion at 4 dpf and imaged axon terminals 4 hours post-conversion (hpc; Fig. 3a ). By 4 hpc, cell body-derived mitochondrial density in the axon terminal was significantly increased in PGC-1α transgenics, confirming that mitochondrial biogenesis in the cell body impacts mitochondrial addition to the axon terminal population ( Fig. 3b, c ). Next, we assessed the density of cell body-derived mitochondria in actr10 mutant axon terminals using the same photoconversion strategy. This demonstrated a 50% reduction in the contribution of cell body-derived mitochondria to the axon terminal population in actr10 mutants compared to wild type ( Fig. 3b, c ). Download figure Open in new tab Figure 3: Cell body-derived mitochondrial density in the axon terminal is reduced in actr10 mutants. (a) Photoconversion strategy. Mitochondrial matrix-localize mEos in the pLLg was locally converted using 405 nm laser. An axon terminal in the mid-trunk (neuromast 3 - NM3) was imaged 4 hpc (b) or every minute for 6h (d). (b) NM3 4 hpc in wild type, actr10 , and transgenic overexpressing (OE) PGC-1α in neurons (Tg(-5kbneurod1:PGC1 α -2A-mRFP) uwd 9 ) . (c) Mean fluorescence intensity of cell body-derived mitochondria (Converted) in the axon terminal population (Steel-Dwass). (d) Representative timepoint of a pLL axon terminal from 6 hr timelapse video (Movies S5 and S6). Cell body-derived mitochondria indicated with white arrowheads. (e, f) Quantification of the number (e, ANOVA) and size (f, Wilcoxon) of converted mitochondria entering the axon terminal. Scale bars = 5 μm. All data are mean ± SEM. Data points represent individual larvae in c and e and individual mitochondria measured from 4 wild type and 3 actr10 axon terminals in f. Reduced cell body derived mitochondrial material in the axon terminal could be due to either fewer mitochondria or smaller mitochondria being transported to the terminal. To differentiate this, we tracked mitochondrial addition to the axon terminal using time-lapse imaging. Mitochondria in the pLL ganglion carrying matrix localized mEos were photoconverted and the axon terminal was imaged every minute for 6 hours (Supplementary Movies 5 and 6). actr10 mutants had both fewer and smaller cell body derived mitochondria entering the terminal per hour ( Fig. 3d-f ). Together, this suggests mitochondrial biogenesis in the cell body contributes to both number and size of new mitochondria transported to the axon terminal. Disrupted mitochondrial fission does not alter mitochondrial biogenesis signatures At the light microscopy level, mitochondria in actr10 mutant pLL cell bodies appear to have a more circular and aggregated morphology, which can be indicative of impaired fission (see Fig. 1b ; 53 , 54 ). To better understand the structure of pLL neuron cell body mitochondria, we used TEM to assess their ultrastructure in actr10 mutants. In wildtype pLL cell bodies, mitochondria were evenly distributed throughout the cytosol (Supplementary Fig. 5a). In actr10 mutants, cell body mitochondrial density and morphology were variable: some cell bodies had fewer but significantly enlarged mitochondria (Supplementary Fig. 5b; 5/12 cell bodies), some showed a more even distribution similar to wildtype (Supplementary Fig. 5c; 1/12 cell bodies), and some showed no visible mitochondria (Supplementary Fig. 5d; 6/12 cell bodies). In contrast, even wildtype cell bodies with little visible cytosolic area still had mitochondria present (Supplementary Fig. 5e; 0/10 cell bodies with no visible mitochondria). On average, mitochondria were significantly larger with a reduced number in actr10 cell bodies (Supplementary Fig. 5f, g). Altered mitochondrial structure could suggest impaired mitochondrial health. However, previous work has shown actr10 mutants have normal mitochondrial health measures in the cell body, and our TEM images show normal cristae even in the enlarged mitochondria (Supplementary Fig. 5b; 37 ). Together, these data show that actr10 mutant mitochondria are larger and rounder than those in wild type siblings, despite not showing altered measures of health and productivity 37 . Altered mitochondrial fission can result in large, circular organelles 53 , 54 . Given the relationship between mitochondrial fission and mitochondrial biogenesis, we asked if disrupted mitochondrial fission in actr10 mutants could underlie disrupted mitochondrial biogenesis 11 , 12 . First, we assessed length of moving and stationary mitochondria in pLL axons in actr10 mutants and wild type siblings as a proxy for fission dynamics. These data showed no change in mitochondrial length, suggesting no major changes in fission or fusion dynamics in the axon (Supplementary Fig. 5h). The cell body mitochondria are too dense for us to directly measure their length or assess fission frequency. Instead, we disrupted mitochondrial fission and assessed measures of mitochondrial biogenesis. To disrupt mitochondrial fission, we expressed a dominant negative form of Drp1 which mimics a constitutively GDP-bound form of the enzyme (Drp1 K38A ; 54 , 55 ). In cultured cells, expression of Drp1 K38A disrupts mitochondrial fission 54 , 55 . When expressed in pLL neurons, Drp1 K38A causes clustering of mitochondria to the perinuclear region of the cell body and reduces mitochondrial density in both wild type and actr10 cell bodies ( Fig. 4a, b ). However, Drp1 K38A expression does not change measures of mitochondrial biogenesis including tfam expression or the number of SSBP1 labeled replicating mtDNA nucleoids ( Fig. 4c-f ). These data argue that loss of fission is not causal in the actr10 mutant mitochondrial biogenesis phenotype. Download figure Open in new tab Figure 4: Disrupting mitochondrial fission does not alter tfam expression or mtDNA replication. (a) Single pLL cell bodies (outlined) expressing mitoTagRFP in wild type and actr10 mutant larvae expressing Drp1 K38A -RFP or RFP (control). (b) Quantification of mitochondrial density (mitochondrial area/cytosolic area; Steel-Dwass). (c) HCR RNA FISH labeling of tfam mRNA in a pLL cell body (outlined). (d) Mean fluorescence intensity of tfam normalized to background (Steel-Dwass). (e) SSBP1 immunostaining in pLL cell body mitochondria, visualized with mitochondria-localized GFP (Mito, magenta; cell outlined). (f) Number of SSBP1 puncta normalized to mitochondrial volume (ANOVA). All data are mean ± SEM. Data points represent individual larvae. MitoTruck mediated disruption of mitochondrial transport inhibits mitochondrial biogenesis Our data demonstrate that inhibiting mitochondrial return to the cell body impairs mitochondrial biogenesis. To test this in another model, we engineered a synthetic tether, similar to one previously described in C. elegans, to constitutively move mitochondria out of the cell body and inhibit their ability to return to the cell body from the distal axon (MitoTruck; 56 ). For this, we tethered mitochondria to a constitutively active kinesin motor domain derived from KIF1A using the OMP25 outer mitochondrial membrane localization sequence ( Fig. 5a ; 57 , 58 ). When expressed in pLL neurons, MitoTruck localizes to mitochondria and ectopically concentrates these organelles to axon ends, demonstrating anterograde transport bias ( Fig. 5b ). Constitutive expression of MitoTruck caused failed axon extension and neuronal death by 4 dpf. To avoid any phenotypes due to failed neuronal health, we analyzed mitochondrial parameters after 24 hours of MitoTruck expression, at 2 dpf. Mitochondria in the cell body are significantly depleted in MitoTruck expressing neuron cell bodies by 2 dpf ( Fig. 5c, d ). This depletion is similar to that observed in actr10 mutants at 4 dpf (mitochondrial density: WT 2 dpf 0.57 ± 0.018, MitoTruck 2 dpf 0.33 ± 0.016, WT 4 dpf 0.55 ± 0.027, actr10 4 dpf 0.26 ± 0.028). Analyses of mtDNA replication and tfam expression demonstrated that MitoTruck significantly decreased these markers of mitochondrial biogenesis ( Fig. 5e-h ). Together our actr10 mutant and MitoTruck analyses demonstrate that inhibiting mitochondrial return to the cell body from the axon is sufficient to impair markers of mitochondrial biogenesis. Download figure Open in new tab Figure 5: MitoTruck expression decreases cell body mitochondrial density and markers of mitochondrial biogenesis. (a) Schematic of the MitoTruck construct. The constitutively active KIF1A motor domain was tethered to mitochondria with the OMP25 outer mitochondrial membrane localization signal (MLS) and visualized with GFP. (b,c) pLL axon (b) and cell body (c; outlined) expressing MitoTruck (green) and MitoTagRFP (Mito, magenta). (d) Quantification of pLL cell body mitochondrial density (mitochondrial area/cytosolic area; Wilcoxon). (e) Representative images of HCR RNA FISH labeling of tfam mRNA in a single pLL cell body (outlined) without and with MitoTruck expression. (f) Mean fluorescence intensity of tfam normalized to background (ANOVA). (g) SSBP1 immunostaining in a single pLL cell body (outlined). Mitochondria visualized with mitochondrially localized TagRFP (Mito, magenta). (h) Number of SSBP1 puncta normalized to mitochondrial volume (ANOVA). Scale bars = 5 μm. All data are mean ± SEM and points represent individual larvae. Estrogen-related receptors bridge mitochondrial transport with transcriptional regulation of mitochondrial biogenesis Our data indicate retrograde transport is required to promote mitochondrial biogenesis through regulation of upstream factors including nuclear gene expression. Several nuclear transcription factors have been identified that bind the promoters of a wide range of mitochondrial genes, allowing for coordinated expression of these genes during biogenesis 10 , 59 , 60 . We hypothesized that a key transcription factor could link mitochondrial retrograde transport to mitochondrial biogenesis. To identify this transcription factor, we performed transcription factor enrichment analysis using significantly downregulated genes from our RNA-seq dataset. This in silico analysis identified Estrogen related receptors (ERRs) as top candidates ( Fig. 6a and Supplementary Fig. 6a). ERRs have been shown to be coactivated by PGC-1α to regulate mitochondrial biogenesis 59 , 61 – 63 . Other transcription factors regulated by PGC-1α such as NRF1/2 or PPARα were not found to be enriched in our analysis, suggesting ERRs specifically may be affected in actr10 mutants. To validate this result, we used gene set enrichment analysis of transcription factor binding targets and identified the ERR binding motif ERR1 enriched near downregulated genes (Supplementary Fig. 6b). Finally, we took a more targeted approach by comparing our list of downregulated genes to ESRRG targets identified by ChIP-seq in cultured neurons 63 . We found significant enrichment for known neuronal ESRRG target genes in our data set ( Fig. 6b ). In contrast, there was only one target gene from the Chip-seq dataset that was significantly upregulated in our dataset (p=0.885, Fisher’s exact test). Together, these in silico analyses suggest ERR-dependent transcription is reduced in actr10 mutants. Download figure Open in new tab Figure 6: ERR activity links mitochondrial transport and mitochondrial biogenesis. (a) Transcription factor enrichment analysis for genes significantly downregulated in actr10 mutants. Plot shows log2(Odds Ratio) vs. log10(adjusted p-value). Top 10 enriched genes based on euclidean distance from log2(OR)/log10(adj. p-value) to the origin are labeled. Box indicates ERR transcription factors identified in the dataset. (b) Overlap between downregulated genes and ESRRG neuronal ChIP-seq targets (Fisher’s exact test). Human homologs of downregulated zebrafish genes were used for transcription factor analyses. Duplicated zebrafish paralogues were only counted once in the analysis. (c) Mitochondria in wild type and actr10 pLL cell bodies (outlined) expressing Esrra or Esrra 4KR . (d) Quantification of mitochondrial density (ANOVAs; Tukey HSD). (e) Mitochondria in wild type and actr10 pLL cell bodies (outlined) with and without resveratrol (+ res.) treatment. (f) Quantification of mitochondrial density (ANOVAs; Tukey HSD). (g) Heat map of mRFP-Esrra or mRFP-Esrra 4KR in wild type and actr10 pLL cell bodies (outlined). (h) Mean fluorescence intensity of nuclear mRFP-Esrra/cytosolic mRFP-Esrra (Steel-Dwass). (i) SSBP1 immunostaining in pLL cell body mitochondria, visualized with mitochondria-localized GFP (Mito, magenta; cell outlined). (j) Number of SSBP1 puncta normalized to mitochondrial volume (ANOVAs; Tukey HSD). (d, f, h, j) Data points represent individual larvae; data are mean ± SEM. Scale bars = 5 μm. Mitochondrial density = mitochondrial area/cytosolic area. If disruption of ERR-dependent transcription underlies decreased mitochondrial biogenesis in actr10 mutant neurons, increasing ERR activity should rescue this defect. To test this, we overexpressed Esrra in pLL neurons and assessed mitochondrial density and biogenesis transcriptional programs. Similar to PGC-1α overexpression (see Supplementary Fig. 4j; 51 ), Esrra overexpression significantly increased tfam expression (Supplementary Fig. 6c, e). Furthermore, Esrra overexpression in actr10 mutants rescues tfam expression to wild type levels (Supplementary Fig. 6d, f). However, overexpression of Esrra was unable to rescue the loss of cell body mitochondrial density in actr10 mutants, suggesting additional levels of ERR regulation are required for optimal mitochondrial biogenesis activation ( Fig. 6c, d ). Activation of ERR-dependent transcription is modulated by ERR or PGC-1α posttranslational modifications (PTMs). We hypothesized PTMs are required for full Esrra activity. The most well studied modifications that activate this pathway are SIRT1-dependent deacetylation of ERR and/or PGC-1α and AMPK-dependent phosphorylation of PGC-1α 64 – 68 . To determine if SIRT1 or AMPK activation were upstream of ERR-dependent mitochondrial biogenesis in neurons, we treated wild type and actr10 mutant zebrafish with resveratrol (activator of SIRT1) or AICAR (activator of AMPK) at concentrations previously optimized for use in zebrafish larvae 64 , 69 , 70 . Resveratrol, but not AICAR, treatment was sufficient to rescue cell body mitochondrial load in actr10 mutant neurons ( Fig. 6e, f and Supplementary Fig. 6g, h), suggesting SIRT1-mediated deacetylation of ERRs may be disrupted in actr10 mutants. SIRT1 has been shown to deacetylate human ESRRA at 4 conserved lysine residues (amino acids 129, 138, 160, and 162) within the DNA-binding domain. This deacetylation can be mimicked by mutating the 4 lysine residues to arginine (4KR), which conserves the basic charge of lysine but prevents acetylation 67 . Overexpression of Esrra 4KR in pLL neurons was able to rescue actr10 cell body mitochondrial density ( Fig. 6c, d ), similar to rescue by resveratrol, suggesting deacetylation is critical for upregulation of mitochondrial biogenesis by Esrra. This was further supported by the localization of Esrra protein. Wild type Esrra did not display a canonical nuclear localization 71 and instead remained largely cytosolic. In contrast, Esrra 4KR displayed strong nuclear localization, suggesting deacetylation is critical for nuclear localization to subsequently activate mitochondrial biogenesis pathways ( Fig. 6g, h ). Further supporting a role for deacetylation in Esrra-dependent mitochondrial biogenesis, overexpression of Esrra 4KR fully rescues SSBP1 punctal density in actr10 mutants ( Fig. 6i, j ). These data demonstrate that ERR deacetylation is critical for regulation of mitochondrial biogenesis in pLL neurons. SIRT1 is a nuclear-localized protein that is activated by elevated NAD+ levels 72 . To determine if NAD+ levels were altered in actr10 nuclei, we used a genetically encoded NAD+ sensor localized to the nucleus 73 . Analysis of steady state NAD+ levels revealed a significant reduction in nuclear NAD+ in actr10 mutants ( Fig. 7a, b ). A significant pool of cellular NAD+ is stored in mitochondria and changes to NAD+/NADH levels in mitochondria can affect the cytosolic/nuclear pool 74 – 76 . We reasoned that altered mitochondrial transport could change local mitochondrial NAD+ levels, leading to a reduction in local SIRT1 activation in the cell body. To test this, we assessed mitochondrial NAD+ in the cell body and axon terminal of pLL neurons by localizing the genetically encoded biosensor to the mitochondria matrix. We found significantly elevated NAD+ levels in axon terminal mitochondria in actr10 mutants while cell body mitochondria have a significant reduction in mitochondrial NAD+ levels compared to wild type controls ( Fig. 7c-e ). Finally, we asked if retrograde transport was responsible for moving mitochondria with higher NAD+ measures from the axon terminal to the cell body. Using live imaging, we assessed mitochondrial NAD+ levels in moving and stationary organelles using the same NAD+ sensor. These experiments demonstrated that retrogradely moving mitochondria have significantly higher NAD+ levels than either stationary or anterogradely moving mitochondria ( Fig. 7f, g ). Together, our data support a model in which the mitochondria with high levels of NAD+ returning to the cell body from the axon elevate local NAD+ levels to activate SIRT1-dependent ERR transcriptional activity necessary for sustained mitochondrial biogenesis in neurons ( Fig. 7h ). We propose that disrupting retrograde mitochondrial transport locks mitochondria with high NAD+ in the axon terminal, blocking this critical signaling step for the positive regulation of mitochondrial biogenesis. Download figure Open in new tab Figure 7: NAD+ levels are altered in actr10 mutants. (a) pLL cell body expressing a nuclear-localized NAD+ sensor. Black outline surrounds single pLL cell body. Dotted blue outline surrounds nucleus. (c) pLL cell body and axon terminal expressing a mitochondrial matrix-localized NAD+ sensor. Black outline surrounds single pLL cell body and axon terminal. Dotted blue outline surrounds mitochondria. (f) Kymograph of pLL axon expressing a mitochondrial matrix-localized NAD+ sensor. (a, c, f) Inverted heat map indicates levels of NAD+. (b, d, e, g) Mean fluorescence intensity of NAD+ sensor normalized to cytosolic RFP (b, d, e) or mitochondrial matrix-localized TagRFP (g). Data inverted to reflect the direction of change in NAD+ levels as fluorescence intensity decreases in the presence of NAD+ (b, d: Wilcoxon; e: ANOVA; g: Steel-Dwass). (h) Model of mitochondrial retrograde transport regulation of mitochondrial biogenesis. Data are mean ± SEM. (b,d, e) Data points represent individual larvae. (g) Data points represent individual mitochondria from 14 wild type larvae. Scale bars = 5 μm. Discussion Mitochondrial population maintenance is essential for neuronal health and function. These organelles are critical for a variety of functions including ATP production and calcium buffering, particularly in axon terminals 1 , 2 . Through these functions, mitochondria accrue damage. Damaged organelles must be replaced through the process of mitochondrial biogenesis. Markers of mitochondrial stress such as an increased AMP/ATP or NAD+/NADH ratio as well as increased cellular calcium can activate mitochondrial biogenesis transcriptional programs in the nucleus 3 – 5 , 60 , 76 – 78 . However, it was unclear how these cellular signals could be transmitted long distances in the neuron to monitor mitochondrial populations in more distal regions of the neuron like the axon terminal. We identified a required role for retrograde return of axonal mitochondria to the cell body to sustain mitochondrial biogenesis transcriptional programs. We show that the return of axonal mitochondria to the cell body stimulates ERR-dependent nuclear gene transcription to regulate expression of mitochondrial genes, mtDNA replication, and ultimately increase mitochondrial biomass in the neuron. This provides a mechanism by which mitochondria in more distal regions of the neuron like the axon terminal can communicate with the nucleus to regulate mitochondrial density and mitochondrial population turnover in neurons. The estrogen-related receptors (ESRRA, ESRRB, and ESRRG) are a family of orphan nuclear receptors first identified due to sequence similarity with estrogen receptors 71 , 79 , 80 . The three ERR proteins bind the ERR response element (ERRE) which is found in the promoter of many mitochondrial genes 59 , 71 . Because of this, increased ERR activity is associated with increased mitochondrial biogenesis in a number of tissues, including neurons 61 , 62 , 73 , 81 . Despite their similarity to estrogen receptors, ERRs do not have the capacity to bind estrogen, but rather are thought to be constitutively active 82 . Their activity can be regulated through expression level, PTMs, or interaction with co-activators like PGC-1α 8 , 9 , 81 . PGC-1α regulates ERR transcription and also interacts with ERRs as a coactivator to induce the transcription of mitochondrial biogenesis genes 8 , 9 , 62 . ERRs serve as mediators between mitochondrial signals and biogenesis transcriptional programs. In non-neuronal cells, signs of mitochondrial depletion or failed health like reduced cellular energy or increased cytosolic calcium have been shown to activate ERR-dependent transcription, either directly or through PGC-1α activation 13 , 83 . These upstream signals of cellular and mitochondrial health act through enzymes that can directly modify ERR and PGC-1α via PTMs including phosphorylation and acetylation. Two of the most well studied upstream regulatory factors are AMPK and SIRT1, which serve as energy sensors for the cell 6 . AMPK is activated by an increased AMP to ATP ratios and can activate mitochondrial biogenesis through PGC-1α phosphorylation 4 , 84 . SIRT1 is activated by an increased NAD+ to NADH ratio and can activate mitochondrial biogenesis through PGC-1α and/or ERR de-acetylation 3 , 67 . AMPK and SIRT1 can be artificially activated through AICAR and resveratrol treatment, respectively, to induce mitochondrial biogenesis 64 , 65 , 69 , 84 , 85 . In our experiments, resveratrol, but not AICAR, treatment was able to rescue actr10 biogenesis defects, suggesting SIRT1-dependent mitochondrial biogenesis is lost. SIRT1 has been shown to deacetylate human ESRRA at four lysine residues to increase ERRA transcriptional activity 67 . Overexpression of an Esrra mutant mimicking deacetylation at these sites was able to rescue markers of mitochondrial biogenesis in actr10 mutants, including mitochondrial density and mtDNA replication. Together, this suggests retrograde transport of axonal mitochondria may stimulate ERR-dependent mitochondrial biogenesis through activation of SIRT1, though we cannot rule out other upstream modulators which could function with SIRT1 in this process. SIRT1 requires NAD+ as a co-substrate for the removal of acetyl groups from lysines, making NAD+ levels a direct regulator of SIRT1 enzymatic activity 72 . Increased NAD+ levels have been shown to increase SIRT1-mediated de-acetylation of PGC-1α, increasing its transcriptional activity 77 , 85 . Therefore, local NAD+ levels are a likely signaling mechanism by which non-neuronal cells are able to sense energy levels and respond with transcriptional regulation of mitochondrial biogenesis. However, it was unclear how the predominantly nuclear-localized SIRT1 could sense and respond to levels of NAD+ in mitochondrial populations spread throughout the expansive dendritic and axonal arbors of neurons 86 . Our data suggests that retrograde transport is required to move mitochondria with high levels of NAD+ to the cell body to stimulate biogenesis. We show retrogradely transported mitochondria have higher NAD+ levels and that the NAD+ levels in axon terminal mitochondria are increased when retrograde transport of mitochondria is impeded. We find that failure to return mitochondria with elevated NAD+ levels to the cell body in actr10 mutants leads to reduced nuclear and cell body mitochondrial NAD+ levels. Based on this, we propose that local SIRT1 activity in the cell body requires increases in the local NAD+ levels which is accomplished by the movement of mitochondria with elevated NAD+ from the axon back to the cell body. Consequent activation of SIRT1 in the cell body then promotes mitochondrial biogenesis through de-acetylation of transcriptional regulators like PGC-1α or ERRs. In support of this, treatment with resveratrol, which circumvents the requirement for NAD+, can rescue mitochondrial density in actr10 mutant cell bodies. It is likely that additional energy sensing pathways work with NAD+/SIRT1 to control mitochondrial biogenesis through additional transcription factor PTMs. Further work on transcription factor PTMs and associated modulatory pathways will clarify the role of energy sensing through NAD+ and other signaling molecules on the control of mitochondrial biogenesis in neurons. In conclusion, our work demonstrates that transport of mitochondria from the axon back to the cell body is a positive regulator of neuronal mitochondrial biogenesis through regulation of ERR-dependent gene transcription. Mitochondria derived from biogenesis in the neuronal cell body are critical for maintaining cell body mitochondrial density as well as supplying the anterogradely transported mitochondria that support the axon terminal mitochondrial population. Unsurprisingly, impaired mitochondrial biogenesis can diminish neuronal health and has been linked to diseases like Parkinson’s and Alzheimer’s diseases 87 – 90 . However, regulatory mechanisms that govern this critical process in the neuron remain poorly understood. Here we show that neurons in zebrafish larvae require mitochondrial retrograde transport for effective mitochondria-nuclear communication to regulate mitochondrial biogenesis through ERRs. This mechanism provides new insight into how neurons regulate mitochondrial population health and density in response to changing demands on the organelle in distal compartments. Funding National Institutes of Health grant R01NS124692 (CMD) National Science Foundation grant DEG-2137424 (AEL) National Institutes of Health grant T32GM007133 (University of Wisconsin-Madison Predoctoral Training Program in Genetics; AEL) University of Wisconsin-Madison Office of the Vice Chancellor for Research (CMD) Wisconsin Alumni Research Foundation (CMD) Author Contributions Conceptualization: AEL, CMD Data acquisition and analysis: AEL, CS, RS, CMD Funding acquisition: AEL, CMD Writing – original draft: AEL Writing – review & editing: CMD Competing interests The authors declare no competing interests. Data and materials availability All data and materials are available upon request from the corresponding author. Materials and Methods Zebrafish husbandry All zebrafish ( Danio rerio ) work was done in accordance with the University of Wisconsin-Madison Institutional Animal Care and Use Committee guidelines. Adult zebrafish were kept at 28°C and embryos were spawned according to established protocols 91 . Embryos and larvae were kept in embryo media (995 μM MgSO4, 154 μM KH2PO4, 42 μM Na2HPO4, pH 7.2, 1.3 mM CaCl2, 503 μM KCl, 15 mM NaCl, 714 μM NaHCO3), maintained at 28°C, and developmentally staged using established methods 92 . All experiments were performed at 4 dpf unless otherwise specified. Sex is not determined at this stage. Zebrafish strains used for these experiments are listed in Table 1. The Tg( hsp70l:EGFP-en.sill ) uwd 12 transgenic was derived using Tol2-mediated transgenesis and the hsp70:eGFP-SILL plasmid as described 93 . Genotyping for actr10 nl 15 35 , p150a y 625 37 , p150b nl 16 35 , and nudc nl 21 42 was done as previously described using the primers in Table 2. Cloning The DNA expression plasmids used in this paper are listed in Table 1. Novel DNA expression constructs were constructed using Gateway cloning 93 or Gibson cloning with oligonucleotides listed in Table 2 94 . 5kbneurod:POLG2-GFP-p2a-mitoTagRFP was created from a human POLG2-GFP template plasmid 12 , 46 . 5kbneurod1:kif1a-egfp-omp25 [“MitoTruck”] was created using the zebrafish outer membrane protein 25 (OMP25) mitochondrial localization sequence (MLS) cloned from 4 dpf zebrafish cDNA. The truncated constitutively active rat ortholog of KIF1A was cloned from pBA-kif1a393-tdTomato-FKBP 95 . 5kbneurod1:cox8a-cox8a-halotag was created by fusing two copies of the cox8a MLS to a HaloTag. 5kbneurod1:esrra-p2a-mRFP was created using the zebrafish esrra sequence derived from 4 dpf zebrafish cDNA and existing p2a and mRFP plasmids 93 . Esrra 4KR expression plasmid was created using consecutive site-directed mutagenesis reactions with the Agilent QuikChange Lightning Site-Directed Mutagenesis Kit. Lysines at amino acid 129, 138, 160, and 162 in the zebrafish ortholog of Esrra were converted to arginines. 5kbneurod1:cox8a-cox8a-NAD-p2a-RFP was created from plasmids containing a double cox8a MLS, a NAD+ biosensor (Addgene plasmid 186791; 96 ) and a p2a-RFP. 5kbneurod1:H2B-NAD-p2a-RFP and 5kbneurod1:cox8a-cox8a-NAD-p2a-mitoTagRFP were created from the 5kbneurod1:cox8a-cox8a-NAD-p2a-RFP plasmid and either a plasmid containing an H2B nuclear localization signal or a p2a-mitoTagRFP.The NAD+ biosensor consists of a circularly permuted Venus fluorescent protein (cpVenus) with a bipartite NAD+-binding domain modeled from bacterial DNA ligase 96 . 5kbneurod1:cox8a-cox8a-paGFP-p2a-mitoTagRFP was created from plasmids containing a double cox8a MLS, a photo-activatable GFP, and a p2a-mitoTagRFP. hsp70l:EGFP-en.sill was created using existing plasmids containing the hsp70 minimal promotor, eGFP, and the 3’ SILL enhancer element that limits expression to the pLL ganglion 93 , 97 . hsp70l:POLG2-GFP-p2a-mitoTagRFP was created from the 5kbneurod:POLG2-GFP-p2a-mitoTagRFP plasmid and the hsp70 minimal promoter. For all expression plasmids except the hsp70 driven constructs, expression was controlled by a 5kb portion of the neurod promotor as previously described 93 , 98 . All novel DNA expression plasmids were sequenced and verified prior to use. Live imaging acquisition and analysis For live imaging of pLL neurons, we used a combination of transient transgenesis and stable transgenic expression. For transient transgenesis, 3-25 pg of plasmid DNA was microinjected into zebrafish zygotes as previously described 35 , 99 , 100 . Plasmids used for these experiments are listed in Table 1. For imaging, zebrafish larvae were sorted to identify larvae with expression in a subset of pLL neuron cell bodies using an AxioZoom V.16 Zeiss microscope. For HaloTag constructs, larvae were treated with Janelia Fluor 635 in embryo media at 1 μM for 1-2 h or 0.1 μM overnight at 28.5°C in the dark. For all live imaging, larvae were anesthetized in 0.02% tricaine, mounted in 1.5% low melt agarose in embryo media, and imaged with an Olympus FV3000 confocal microscope with a 40x (NA 1.25) silicone oil objective. Optimal interslice interval was used for all imaging. Larval TgBAC( neurod:egfp ) nl 1 ( Fig. 1a ) and Tg(hsp70:eGFP-SILL ) uwd12Tg (Supplementary Fig. 1a) transgenic zebrafish images were created by stitching overlapping regions of the larvae imaged using the Olympus FV3000 with a 10X objective using the pairwise stitching plugin in ImageJ 101 , 102 . Non-linear adjustment to midtones to show the full nervous system was done using the levels function in Adobe Photoshop. For TgBAC( neurod:egfp ) nl 1 , a brightfield image of the full larva was taken using the AxioZoom V.16 Zeiss microscope with a Zeiss Axio 506 color camera and overlaid using Adobe Photoshop with opacity adjusted to 10%. For Tg(hsp70:eGFP-SILL ) uwd12Tg , brightfield images from the Olympus FV3000 were stitched and overlaid with an opacity of 30%. To quantify mitochondrial density in the pLL cell body and axon terminal, area was measured from standard deviation z-projections in ImageJ. Mitochondrial area was measured using a manually applied threshold while cytosolic area was measured by manual tracing or applied threshold of a cytosolic fill. Mitochondrial density was calculated as mitochondrial area divided by cytosolic area. To quantify mitochondrial or cell volume, the 3D objects counter was used in ImageJ using a manually applied threshold 102 . For POLG2 axon terminal analysis, 5kbneurod:POLG2-GFP-p2a-mitoTagRFP was transiently expressed in pLL neurons by zygotic microinjection as described 100 and terminal regions were imaged as described above. POLG2+ puncta were manually counted and mitochondrial volume was quantified as described above. Mitochondrial axonal transport analyses were done as described previously 100 . Briefly, transient transgenesis of 5kbneurod1:mitotagRFP was used to visualize mitochondria in pLL axons. 30–100 μm lengths of axon were imaged as a single z -plane at a rate of 300 ms per frame for 1000 frames. Kymographs were generated using the ImageJ Multi Kymograph function 102 . Anterograde, retrograde, and bidirectional mitochondria number were quantified using kymographs while stationary mitochondria number was quantified by visual analysis of transport videos. Mitochondrial length was manually measured from transport movies using the line selection tool in ImageJ. For NAD+ quantification, NAD sensor constructs were transiently expressed in pLL neurons by zygotic microinjection. For single timepoint imaging, larvae were mounted live as described above and cell body and axon terminal regions were imaged at 4 dpf with consistent laser power and detector gain for all images. Transport analysis was done as described above with sequential imaging of cpVenus and RFP channels 35 , 100 . For cell body and axon terminal mitochondria analysis ( 5kbneurod1:cox8a-cox8a-NAD-p2a-RFP ), cell body and axon terminal regions were isolated using the mRFP cytosolic signal as a mask and the image subtraction function in ImageJ 102 . cpVenus and mRFP mean fluorescence intensity were then quantified from sum projections of the isolated region. For nuclear analysis ( 5kbneurod1:H2B-NAD-p2a-RFP ), sum projections were created for cpVenus and mRFP. Nuclear cpVenus mean fluorescence intensity was quantified by manual outlining of nucelar area. mRFP mean fluorescence intensity was quantified by manual outlining of cytosolic area. For transport analysis ( 5kbneurod1:cox8a-cox8a-NAD-p2a-mitoTagRFP) , mitochondria were identified as moving anterogradely, retrogradely, or stationary by visual analysis of transport videos. cpVenus and RFP mean fluorescence intensity were quantified by manual outlining of individual mitochondria. For all analyses, mean fluorescence intensity of cpVenus was normalized to mean fluorescence intensity of RFP to control for differences in expression level. NAD+ binding to the bipartite NAD+-binding domain in the biosensor decreases cpVenus fluorescence intensity in a dose-dependent manner 96 . The images in Figure 7 were generated using the fire lookup table in ImageJ and the images/data are inverted to appropriately reflect the direction of change in NAD+ levels. Drug treatment Resveratrol and AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) stocks (200 mM and 27.5 mM respectively) were diluted in embryo media to concentrations previously shown to be effective in larval zebrafish (Resveratrol 300 μM, AICAR 1 mM; 70 ). At 3 dpf, transient transgenic larval zebrafish expressing cytosolic GFP and mitochondrially localized tagRFP (see Table 1 for DNA constructs) were placed in a 6 well plate and incubated overnight (18-24 h) in drug or equal amount of drug solvent (DMSO for resveratrol, sterile H 2 O for AICAR) in embryo media. After treatment, larvae were mounted live, pLL neuronal cell bodies were imaged, and mitochondrial density analyzed as described above. Heat shock-induced expression of Drp1 K38A and POLG2-GFP For DRP-1 analysis, Hsp701:Drp1K38A-mRFP 35 or 5kbneurod1:RFP 35 (control) was transiently expressed with 5kbneurod1:mito-eGFP in pLL neurons by zygotic microinjection as described 100 . At 4 dpf, larvae of both groups were placed in PCR strip tubes (2-3 larvae per tube) with approximately 200 μl of embryo media. Larvae were incubated at 37°C for 1 hour in a PCR machine (BioRad) then returned to 28°C. Larvae were fixed in 4% paraformaldehyde 7 hours post-heat shock for subsequent HCR RNA FISH or immunolabeling as described below. For POLG2-GFP cell body puncta analysis, Hsp701:POLG2-GFP-p2a-mitoTagRFP was transiently expressed in pLL neurons by zygotic microinjection as described 100 . At 4 dpf, embryo media was pre-heated to 37°C in 50 mL conical tubes. Larvae were transferred to the pre-heated embryo media in 50 mL conical tubes and kept at 37°C for 1 hour. Larvae were then returned to 28°C media. Larvae were fixed in 4% PFA/0.25% Triton X-100 7 hours post-heat shock for subsequent imaging. Fixed larvae were mounted between glass slides and #1.5 coverslip with Fluoromount mounting medium prior to imaging with an Olympus FV3000 confocal microscope with a 60x (NA1.42) oil objective. Punctal quantification was done as described above for axon terminals. Nuclear localization analysis 5kbneurod1:mRFP-esrra or 5kbneurod1:mRFP-esrra 4KR was transiently expressed in pLL neurons by zygotic microinjection as described 100 . At 4 dpf, larvae were fixed in 4% PFA/0.25% Triton X-100 and co-incubated with DAPI (1:500) at 4°C overnight. Larvae were imaged as described above for cell body POLG2-GFP. DAPI was used to mask out nuclear RFP signal. Mean fluorescence intensity was then quantified from sum projections of the nuclear and cytosolic regions. Photolabeling 5kbneurod1:cox8a-cox8a-paGFP-p2a-mitoTagRFP was transiently expressed in pLL neurons by zygotic microinjection as described 100 . Cell body or axon terminal mitochondrial-matrix localized PA-GFP was specifically photoactivated by illuminating with a 405 nm laser using a region of interest (ROI) defined around the structure. For each larva, a pre-activation image was taken (488 nm and 561 nm lasers), the region was scanned for 1 to 5 seconds with 1% 405 nm laser power using the stimulation function for conversion, and a post-activation image (488 nm and 561 nm lasers) was taken to confirm activation. The axon terminal was then re-imaged at set timepoints post-conversion. Due to transient expression, PA-GFP was not expressed in every axon terminal. To reduce variability due to differences in axon length, only axon terminals innervating sensory organs in the center of the trunk (neuromasts 2, 3, or 4) were used for analysis. PA-GFP and MitoTagRFP mitochondrial area were quantified as described earlier. “Proportion original mitochondria” for terminal activation and “proportion of cell body-derived mitochondria” for cell body activation was quantified as PA-GFP area/MitoTagRFP area (see Figure S3). Photoconversion of mitochondrially localized mEos was performed using the stable transgenic Tg(-5kbneurod1:mito-mEos) y 586 Tg as previously described 37 . The pLL ganglion was specifically converted by illuminating with a 405 nm laser through a z-stack of the entire ganglion with regional restriction using a region of interest (ROI) defined around this structure. For each larva, a preconversion image was taken (488 nm and 561 nm lasers), the stack was scanned with 1% 405 nm laser power for conversion, and a postconversion image (488 nm and 561 nm lasers) was taken to confirm complete green to red conversion of mEos in pLL cell bodies. For 4 hour timepoint analysis, the axon terminal of a sensory organ in the center of the trunk (NM3) was imaged 4 hours post pLL ganglion conversion. All microscope settings were kept consistent for all images. For axon terminal analysis, the red and green channels were merged and a mask was created by thresholding a standard deviation projection of the axon terminal z -stack. The mask was applied to a sum projection of the red channel and mean fluorescence was quantified. For timelapse analysis, a z-stack through an ROI surrounding NM3 was scanned minute for 6 hours. Image analysis was performed by manually scanning through standard deviation z-stack projections from the time series. Cell body derived (red photoconverted) mitochondria were counted and volume measured using the 3D objects counter in ImageJ. In situ Hybridization and Hybridization Chain Reaction RNA Fluorescent In situ Hybridization (HCR RNA FISH) Colorimetric in situ hybridization was done according to established protocols 99 , 103 . To generate probes for in situ hybridization, we amplified 200-600 base pair regions of relevant cDNA within the open reading frames of genes of interest to use as a template for probe synthesis ( 104 ; Table 2). Zebrafish were fixed overnight in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) at 4°C. Fixed larvae were washed in PBS/0.1% Tween-20 and dehydrated in a methanol series. Larvae were stored in methanol at -20°C. Larvae were rehydrated with PBS/0.1% Tween-20 in a reverse methanol series. Larvae were treated with 10 μg/ml proteinase K in PBS/0.1% Tween-20 for 15 minutes then immediately fixed for 20 min in 4% PFA at room temperature (RT). Larvae were washed with PBS/0.1% Tween-20 then prehybridized for 1-2 hours at 65°C. Larvae were incubated overnight at 65°C with probe diluted 1:200 in hybridization buffer (10 mM citric acid, 0.1% Tween-20, 50 μg/ml heparin, 5× saline sodium citrate (SSC), 50% formamide). Larvae were washed at 65°C with a hybridization buffer-SSC buffer series (2:1 buffer:2x SSC 15 min; 1:2 buffer:2x SSC 15 min; 2x SSC 15 min; 0.2x SSC 30 min twice). Larvae were then washed at RT with 2:1 and then 1:2 0.2x SSC:PBS/0.1% Tween-20 for 5 min. Larvae were blocked in block solution (2% goat serum, 10% bovine serum albumin in PBS/0.1% Tween-20) for 2 hours at RT, and then sheep anti-digoxigenin (1:10,000) in block solution overnight at 4°C. Larvae were washed 6 times 15 min in PBS/0.1% Tween-20, 3 x 5 min in coloration buffer (100 mM Tris–HCl pH 9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20), then stained with 0.45% nitro blue tetrazolium, 0.35% 5-bromo-4-chloro-3-indolyl phosphate in a coloration buffer in dark until color development. Larvae were washed with PBS/0.1% Tween-20, fixed for 20 min in 4% PFA at RT, dehydrated in a methanol series, and rehydrated for imaging and sorting of RNA expression. WT and actr10 mutant zebrafish were kept in the same tube for processing, then blindly sorted based on RNA expression into two groups (high and low expressing). Representative larvae were mounted in 1.5% low melt agarose in embryo media and imaged with a Zeiss Axio Imager.M2 microscope with Zeiss Axio 506 color camera. Sorted larvae were genotyped using the oligonucleotides in Table 2. HCR RNA FISH was performed as described previously 51 . An mRNA transcript-specific probe set for each gene of interest was designed by Molecular Instruments (Table 3). Larvae fixation, methanol dehydration/rehydration, and Pro K treatment were performed as described for colorimetric in situ hybridization with modification in the Pro K treatment for larvae less than 4 dpf. 2 dpf larvae were treated with Pro K for 5 min, 3 dpf for 10 min, and 4 dpf for 15 min, followed by post-fixation. Larvae were incubated with a manufacturer-supplied hybridization buffer at 37°C for 1 h and then incubated in 4 nM ( pgc1a and tfam ) or 16 nM (others) probe in hybridization buffer at 37°C overnight. Larvae were washed with a manufacturer-supplied wash buffer and then SSC/0.1% Tween-20. Then, larvae were incubated in a manufacturer-supplied amplification buffer at RT for 30 min, during which manufacture-supplied reporter-labeled fluorescent hairpins were individually heated at 95°C and then cooled to RT for 30 min in the dark. Larvae were placed in fresh amplification buffer with 60 nM hairpins. B1 probes were labeled with 546 nm wavelength hairpins while B2 probes were labeled with 647 nm wavelength hairpins combined in the same tube. Larvae were incubated overnight in the dark at RT. Larvae were mounted between glass slides and #1.5 coverslip with Fluoromount mounting medium prior to imaging with an Olympus FV3000 confocal microscope with a 60x (NA1.42) oil objective. For each probe, laser power and detector gain were kept consistent for all images To quantify HCR signal in the whole pLL ganglion in TgBAC(neurod:eGFP) nl 1 larvae, a mask was created using the GFP channel threshold using the default auto thresholding algorithm in ImageJ and the image subtraction function was used to remove non-neuronal signal. After using the mask to exclude signal outside the pLLg, a sum projection of the probe signal was created and mean fluorescence intensity was measured. To quantify HCR signal in single cell bodies, a substack was created around the cell of interest and a sum intensity z-projection of the probe signal was created. The cell body was manually outlined and mean fluorescence intensity of the probe signal within the cell body was measured. To quantify background signal, a region outside the pLLg or cell body was selected and the sum intensity of probe signal was measured. Background mean signal was subtracted from pLL mean signal to get the normalized mean fluorescence intensity. Immunolabeling For SSBP1 and TFAM immunolabeling, transient transgenesis of either 5kbneurod1:mito-eGFP or 5kbneurod1:mitotagRFP was used to localize pLL cell body mitochondria. Larvae were fixed in 4% PFA/0.25% Triton X-100 at 4°C overnight then incubated in water at RT overnight. Larvae were kept in block (0.1% Triton X-100, 1% dimethyl sulfoxide, 0.02% sodium azide, 0.5% bovine serum albumin, 5% goat serum) at RT for 2h, then incubated with antibody in block overnight at 4°C. Antibodies were validated by western blot (Supplementary Fig. 1m). Larvae injected with 5kbneurod1:mito-eGFP were incubated with chicken anti-GFP (1:2000) to amplify signal. Larvae were incubated with either rabbit anti-SSBP1 (1:500) or rabbit anti-TFAM (1:100). Larvae were then washed in PBS/0.1% Triton X-100 before incubation in goat anti-chicken AlexaFluor 488 (1:1000) and goat anti-rabbit AlexaFluor 568 (1:1000) or goat anti-rabbit AlexaFluor 647 (1:1000) at 4°C overnight. Larvae were washed in PBS/0.1% Triton X-100 prior to imaging as described for HCR. For analysis, a mask was created from the mitochondrial channel using the default auto thresholding algorithm in ImageJ and the image subtraction function was used to remove signal outside the pLL cell body. For TFAM quantification, the 3D objects counter function in ImageJ and manual thresholding was used to quantify volume of TFAM puncta and mitochondria (mitochondrial volume used for normalization). For SSBP1 quantification, SSBP1 puncta were manually counted and normalized to mitochondrial volume. Transmission Electron Microscopy 4 dpf wild type and actr10 mutant larvae samples were dissected by removing the trunks posterior to the pLLg and then fixed in Karnovsky’s fixative (2.5% glutaraldehyde/2.0% formaldehyde in 0.1M NaPO4 buffer (PB), pH=7.2) at 4°C overnight. All remaining steps were performed on the specimens in glass scintillation vials on a rotator unless specified. The fixative was removed and the samples washed 5 x 5 min in PB at RT. Post-fixation was performed on the samples in 1% OsO4 in PB for 1 hour at RT. The samples were again washed in PB, 7 x 5 minutes at RT. Dehydration was performed with a graded series of EtOH (35%, 50%, 70%, 80%, 90% 5 x 5 minutes, 95% for 10 minutes, 100% for 3 x 10 minutes) at RT. Specimens were transitioned from EtOH by rinsing 2 x 7 minutes in acetone at RT. Samples were infiltrated and embedded in EMBed 812 resin (EMS Hatfield; PA 19440) using a graded series of 2:1 Acetone / EMBed812 1 hour RT, 1:1 Acetone / EMBed812 overnight RT, 1:2 Acetone / EMBed812 2 hours RT. 100% EMBed 812 resin infiltrations continued in uncapped vials for 1 hour at 65°C in a laboratory microwave, with a fresh change of resin for 1 hour at 75°C. Final embedding was done in EMBed 812 resin in aluminum weighing dishes placed in a 60°C drying oven and allowed to polymerize for 2 days. Polymerized samples had the aluminum dishes stripped, with the target specimen area sawed out with a jewelers saw and glued to blank polymerized stubs of EMBed 812 resin. Specimens were sectioned on a Reichert-Jung Ultracut E ultra-microtome with sections collected on formvar coated, Cu 2×1 slot grids. Grids were post-stained in uranyl acetate and Reynolds lead citrate and viewed on a Philips CM120 at 80kV. Images were collected on a BioSprint12 digital camera. One section of the pLLg was imaged for a WT and actr10 mutant larvae. All cells visible within the section were analyzed. Images were calibrated in ImageJ and analyzed by manual counting of mitochondria and manual outlining for mitochondrial, cell, and nuclear area. Cytosolic area was calculated as cell area minus nuclear area. Western blot Western blot of TFAM and SSBP1 protein was done as described previously 35 . Protein lysates of 4 dpf wildtype larvae were run on a 12% sodium dodecyl sulfate polyacrylamide electrophoresis gel. After transfer to the polyvinylidene difluoride membrane, the blots were blocked in 5% nonfat dry milk in 1X PBS/0.1% Tween for 1–4 hours before incubation with anti-TFAM (1:1000) or anti-SSBP1 (1:500) overnight in block at 4°C. Membranes were then washed in 1X PBS, 0.1% Tween20 and incubated with secondary antibody conjugated to horseradish peroxidase for 90 min at RT. The blot was developed with a Western Sure Chemiluminescent Substrate (LiCor) on a C-DiGit Blot Scanner. Embryo dissociation and fluorescence activated cell sorting (FACS) 20-30 4 dpf Tg(hsp70:eGFP-SILL) uwd 12 larvae were collected in 1.5 ml microcentrifuge tubes. Embryo media was removed and larvae were incubated in 100 μL sterile, calcium-free ringer’s solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPES pH 7.0) at RT for 5 min. 600 μL of sterile protease solution (0.25% trypsin, 1 mM EDTA, pH 8.0, PBS) was warmed to 28°C and 27 μl of Collagenase P (100 mg/mL in HBSS) was added to the tube and mixed. Larvae were incubated at 28°C. Every 30s, larvae were homogenized for 30s by pipetting first with a P1000 pipette until partially dissociated, then with a P200 pipette until fully dissociated. Once dissociated, 100 μl of sterile stop solution (30% calf serum, 6 mM CaCl2, PBS) was added and mixed. The cells were spun down at 350xg at 4°C for 5 min. The supernatant was removed and replaced with 500 μl sterile suspension solution (1% FBS, 0.8 mM CaCl2, 50 U/mL penicillin, 0.05 mg/mL streptomycin, DMEM) chilled to 4°C. Cells were spun at 350xg at 4°C for 5 min. The supernatant was removed and 500 μl of chilled sterile suspension solution was used to resuspend cells. Resuspended cells were passed through a 40 μm cell strainer into FACS tube and stored on ice until sorting. Immediately prior to sorting, DAPI (1:1000) was added to sort out dead cells. GFP+ DAPI-cells were sorted on a BD FACSAria™ III Cell Sorter with a 100 μm nozzle into 500 μL TRIzol LS in a 1.5 mL microcentrifuge tube. Bulk RNA sequencing and analysis Sorted pLL neurons were pooled into 3 replicates each for wild type and actr10 samples. RNA was isolated using TRIzol. The SMART-Seq® v4 Ultra® Low Input RNA Kit was used for poly(A)+ selection, cDNA synthesis and amplification, followed by Illumina 150-bp paired-end sequencing performed by Azenta Life Sciences (South Plainfield, NJ). Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36 105 . The trimmed reads were mapped to the Danio rerio GRCz10.89 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b 106 . Unique gene hit counts were calculated by using FeatureCounts from the Subread package v.1.5.2 107 . Only unique reads that fell within exon regions were counted. Using DESeq2 108 , a comparison of gene expression between WT and actr10 samples was performed. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value 1 were called as differentially expressed genes for each comparison. Alignment and differential expression analysis was performed by Azenta Life Sciences (South Plainfield, NJ). KEGG pathway enrichment was performed for genes that were called as significantly upregulated or significantly downregulated using the ShinyGO (v0.82) webserver ( 84-86 ). Danio rerio gene IDs were used as input and background genes were defined as all genes that were detectable in the RNA-sequencing dataset. Default parameters were maintained. KEGG pathway plots were downloaded directly from the ShinyGO webserver. Gene Set Enrichment Analysis (GSEA) for differentially expressed genes was performed using the clusterProfiler R package with all gene ontology terms included, using the genome-wide annotation for Zebrafish (org.Dr.eg.db v3.19.1; 109 ). Zebrafish gene IDs were converted to their closest human gene orthologues using the g:Profiler g:Orth webserver 110 . Fisher’s exact test enrichment analysis was performed using the fisher.test function in R to test overlap between significantly upregulated or significantly downregulated genes and either the MitoCarta database or the ESRRG ChipSeq targets 49 , 63 . Transcription factor enrichment analysis was performed using the TFEA.ChIP webserver which uses information derived from the ChIP-seq datasets available from the ENCODE project, GEO Datasets, GeneHancer, ReMap 2018, and ReMap 2022 databases 111 . The significantly downregulated genes were used with background genes defined as all genes that were detectable in the RNA-sequencing dataset. Wilcoxon rank-sum was used for transcription factor ranking. Over-representation analysis of transcription factor target motifs was performed using the Webgestalt webserver with the functional database identified as ‘‘network: transcription factor target” 112 . The significantly downregulated genes were used with background genes defined as all genes that were detectable in the RNA-sequencing dataset. Default parameters were maintained. Volcano plots of differentially expressed genes and transcription factor enrichment were created using the ggplot2 R package 113 . Experimental design, statistical analysis, and figure assembly All image analysis was performed using ImageJ 102 . Statistical analysis was performed using JMP18 and data plots made using GraphPad Prism 10, unless indicated otherwise in the method section. Sample size estimated based on previous work and determined by power analyses. Imaging and analysis done blind to genotype, allowing inherent randomization and blinding to experimental condition. All experiments were replicated no less than two times (experimental replicates) with sample size for each experiment representing biological replicates. Statistical tests/post hoc analyses and other statistical details for each data set are indicated in the text or figure legends. For parametric data, ANOVAs were used with Tukey HSD post-hoc contrasts for multiple pairwise comparisons within a dataset. For nonparametric data, single comparisons were done using Wilcoxon/Kruskal-Wallis analysis, multiple comparisons using the Steel-Dwaas test. Images were created using ImageJ and linear adjustments to brightness/contrast were made equally for images being compared. Figures were compiled in Adobe Illustrator and supplemental movies were handled in Adobe After Effects. Acknowledgments We would like to acknowledge members of the Drerup lab and Dr. Samantha Lewis for thoughtful discussions of the work and critique of the manuscript. The constitutively active KIF1A motor domain construct was provided by Dr. Marvin Bentley. We would like to thank the University of Wisconsin Carbone Cancer Center Flow Cytometry Laboratory, supported by P30 CA014520, for use of its facilities and assistance with fluorescence activated cell sorting. We would like to thank Randall Massey and the University of Wisconsin School of Medicine and Public Health Electron Microscopy Facility for assistance with Transmission Electron Microscopy. Funder Information Declared National Institute of Neurological Disorders and Stroke , R01NS124692 U.S. National Science Foundation , DEG-2137424 National Institutes of Health , T32GM007133 University of Wisconsin-Madison Office of the Vice Chancellor for Research Wisconsin Alumni Research Foundation Footnotes We have revised some of the text to clarify ideas and add new data. We have also added a significant amount of new data in both the figures and supplement. References 1. ↵ Suomalainen , A. , and Nunnari , J . ( 2024 ). Mitochondria at the crossroads of health and disease . Cell 187 , 2601 – 2627 . doi: 10.1016/j.cell.2024.04.037 . OpenUrl CrossRef PubMed 2. ↵ Misgeld , T. , and Schwarz , T.L . ( 2017 ). Mitostasis in Neurons: Maintaining Mitochondria in an Extended Cellula r Architecture . Neuron 96 , 651 – 666 . doi: 10.1016/j.neuron.2017.09.055 . OpenUrl CrossRef PubMed 3. ↵ Gerhart-Hines , Z. , Rodgers , J.T. , Bare , O. , Lerin , C. , Kim , S.-H. , Mostoslavsky , R. , Alt , F.W. , Wu , Z. , and Puigserver , P . ( 2007 ). Metabolic control of muscle mitochondrial function and fatty acid oxid ation through SIRT1/PGC-1α . The EMBO Journal 26 , 1913 – 1923 . doi: 10.1038/sj.emboj.7601633 . OpenUrl Abstract / FREE Full Text 4. ↵ Jäger , S. , Handschin , C. , St.-Pierre , J ., and Spiegelman , B.M. ( 2007 ). AMP-activated protein kinase (AMPK) action in skeletal muscle via dire ct phosphorylation of PGC-1α . Proceedings of the National Academy of Sciences 104 , 12017 – 12022 . doi: 10.1073/pnas.0705070104 . OpenUrl Abstract / FREE Full Text 5. ↵ Ojuka , E.O. , Jones , T.E. , Han , D.H. , Chen , M. , and Holloszy , J.O . ( 2003 ). Raising Ca 2+ in L6 myotubes mimics effects of exercise on mitochondrial biogenesis in muscle . The FASEB Journal 17 , 675 – 681 . doi: 10.1096/fj.02-0951com . OpenUrl CrossRef PubMed Web of Science 6. ↵ Cantó , C. , Gerhart-Hines , Z. , Feige , J.N. , Lagouge , M. , Noriega , L. , Milne , J.C. , Elliott , P.J. , Puigserver , P. , and Auwerx , J . ( 2009 ). AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity . Nature 458 , 1056 – 1060 . doi: 10.1038/nature07813 . OpenUrl CrossRef PubMed Web of Science 7. ↵ Scarpulla , R.C . ( 2011 ). Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network . Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 1813 , 1269 – 1278 . doi: 10.1016/j.bbamcr.2010.09.019 . OpenUrl CrossRef PubMed Web of Science 8. ↵ Schreiber , S.N. , Emter , R. , Hock , M.B. , Knutti , D. , Cardenas , J. , Podvinec , M. , Oakeley , E.J. , and Kralli , A . ( 2004 ). The estrogen-related receptor α (ERRα) functions in PPARγ coactivator 1α (PGC-1α)-induced mitochondrial biogenesis . Proceedings of the National Academy of Sciences 101 , 6472 – 6477 . doi: 10.1073/pnas.0308686101 . OpenUrl Abstract / FREE Full Text 9. ↵ Schreiber , S.N. , Knutti , D. , Brogli , K. , Uhlmann , T. , and Kralli , A . ( 2003 ). The Transcriptional Coactivator PGC-1 Regulates the Expression and Act ivity of the Orphan Nuclear Receptor Estrogen-Related Receptor α (ERRα) . Journal of Biological Chemistry 278 , 9013 – 9018 . doi: 10.1074/jbc.M212923200 . OpenUrl Abstract / FREE Full Text 10. ↵ Virbasius , J.V. , and Scarpulla , R.C . ( 1994 ). Activation of the human mitochondrial transcriptionfactor A gene by nuclear respiratory factors: a potential regulatory linkbetween nuclear and mitochondrial gene expression in organellebiogenesis . Proceedings of the National Academy of Sciences 91 , 1309 – 1313 . doi: 10.1073/pnas.91.4.1309 . OpenUrl Abstract / FREE Full Text 11. ↵ Kleele , T. , Rey , T. , Winter , J. , Zaganelli , S. , Mahecic , D. , Perreten Lambert , H. , Ruberto , F.P. , Nemir , M. , Wai , T. , Pedrazzini , T. , and Manley , S . ( 2021 ). Distinct fission signatures predict mitochondrial degradation or biogenesis . Nature 593 , 435 – 439 . doi: 10.1038/s41586-021-03510-6 . OpenUrl CrossRef PubMed 12. ↵ Lewis , S.C. , Uchiyama , L.F. , and Nunnari , J . ( 2016 ). ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells . Science 353 , aaf5549. doi: 10.1126/science.aaf5549 . OpenUrl Abstract / FREE Full Text 13. ↵ Cardanho-Ramos , C. , and Morais , V.A . ( 2021 ). Mitochondrial Biogenesis in Neurons: How and Where . International Journal of Molecular Sciences 22 , 13059 . doi: 10.3390/ijms222313059 . OpenUrl CrossRef PubMed 14. ↵ Davis , A.F. , and Clayton , D.A . ( 1996 ). In situ localization of mitochondrial DNA replication in intact mammalian cells . Journal of Cell Biology 135 , 883 – 893 . doi: 10.1083/jcb.135.4.883 . OpenUrl Abstract / FREE Full Text 15. ↵ Schultz , R.A. , Swoap , S.J. , McDaniel , L.D. , Zhang , B. , Koon , E.C. , Garry , D.J. , Li , K. , and Williams , R.S . ( 1998 ). Differential Expression of Mitochondrial DNA Replication Factors in Mammalian Tissues . Journal of Biological Chemistry 273 , 3447 – 3451 . doi: 10.1074/jbc.273.6.3447 . OpenUrl Abstract / FREE Full Text 16. ↵ Amiri , M. , and Hollenbeck , P.J . ( 2008 ). Mitochondrial biogenesis in the axons of vertebrate peripheral neurons . Dev Neurobiol 68 , 1348 – 1361 . doi: 10.1002/dneu.20668 . OpenUrl CrossRef PubMed Web of Science 17. Cioni , J.M. , Lin , J.Q. , Holtermann , A.V. , Koppers , M. , Jakobs , M.A.H. , Azizi , A. , Turner-Bridger , B. , Shigeoka , T. , Franze , K. , Harris , W.A. , and Holt , C.E . ( 2019 ). Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons . Cell 176 , 56 – 72 e15. doi: 10.1016/j.cell.2018.11.030 . OpenUrl CrossRef PubMed 18. Kuzniewska , B. , Cysewski , D. , Wasilewski , M. , Sakowska , P. , Milek , J. , Kulinski , T.M. , Winiarski , M. , Kozielewicz , P. , Knapska , E. , Dadlez , M. , et al. ( 2020 ). Mitochondrial protein biogenesis in the synapse is supported by local translation . EMBO Rep 21 , e48882 . doi: 10.15252/embr.201948882 . OpenUrl CrossRef PubMed 19. ↵ Van Laar , V.S. , Arnold , B. , Howlett , E.H. , Calderon , M.J. , St Croix , C.M. , Greenamyre , J.T. , Sanders , L.H. , and Berman , S.B . ( 2018 ). Evidence for Compartmentalized Axonal Mitochondrial Biogenesis: Mitochondrial DNA Replication Increases in Distal Axons As an Early Response to Parkinson’s Disease-Relevant Stress . J Neurosci 38 , 7505 – 7515 . doi: 10.1523/JNEUROSCI.0541-18.2018 . OpenUrl Abstract / FREE Full Text 20. ↵ Cardanho-Ramos , C. , Simoes , R.A. , Wang , Y.Z. , Faria-Pereira , A. , Bomba-Warczak , E. , Craessaerts , K. , Spinazzi , M. , Savas , J.N. , and Morais , V.A . ( 2024 ). Local mitochondrial replication in the periphery of neurons requires the eEF1A1 protein and the translation of nuclear-encoded proteins . iScience 27 , 109136 . doi: 10.1016/j.isci.2024.109136 . OpenUrl CrossRef 21. ↵ Hollenbeck , P.J. , and Saxton , W.M . ( 2005 ). The axonal transport of mitochondria . J Cell Sci 118 , 5411 – 5419 . doi: 10.1242/jcs.02745 . OpenUrl Abstract / FREE Full Text 22. ↵ Pilling , A.D. , Horiuchi , D. , Lively , C.M. , and Saxton , W.M . ( 2006 ). Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons . Mol Biol Cell 17 , 2057 – 2068 . doi: 10.1091/mbc.e05-06-0526 . OpenUrl Abstract / FREE Full Text 23. Saxton , W.M. , and Hollenbeck , P.J . ( 2012 ). The axonal transport of mitochondria . J Cell Sci 125 , 2095 – 2104 . doi: 10.1242/jcs.053850 . OpenUrl Abstract / FREE Full Text 24. ↵ Schnapp , B.J. , and Reese , T.S . ( 1989 ). Dynein is the motor for retrograde axonal transport of organelles . Proc Natl Acad Sci U S A 86 , 1548 – 1552 . doi: 10.1073/pnas.86.5.1548 . OpenUrl Abstract / FREE Full Text 25. ↵ Canty , J.T. , Hensley , A. , Aslan , M. , Jack , A. , and Yildiz , A . ( 2023 ). TRAK adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport . Nat Commun 14 , 1376 . doi: 10.1038/s41467-023-36945-8 . OpenUrl CrossRef PubMed 26. Glater , E.E. , Megeath , L.J. , Stowers , R.S. , and Schwarz , T.L . ( 2006 ). Axonal transport of mitochondria requires milton to recruit kinesin heavy chain and is light chain independent . J Cell Biol 173 , 545 – 557 . doi: 10.1083/jcb.200601067 . OpenUrl Abstract / FREE Full Text 27. Stowers , R.S. , Megeath , L.J. , Gorska-Andrzejak , J. , Meinertzhagen , I.A. , and Schwarz , T.L . ( 2002 ). Axonal transport of mitochondria to synapses depends on milton, a novel Drosophila protein . Neuron 36 , 1063 – 1077 . doi: 10.1016/s0896-6273(02)01094-2 . OpenUrl CrossRef PubMed Web of Science 28. Guo , X. , Macleod , G.T. , Wellington , A. , Hu , F. , Panchumarthi , S. , Schoenfield , M. , Marin , L. , Charlton , M.P. , Atwood , H.L. , and Zinsmaier , K.E . ( 2005 ). The GTPase dMiro is required for axonal transport of mitochondria to Drosophila synapses . Neuron 47 , 379 – 393 . doi: 10.1016/j.neuron.2005.06.027 . OpenUrl CrossRef PubMed Web of Science 29. Fenton , A.R. , Jongens , T.A. , and Holzbaur , E.L.F . ( 2021 ). Mitochondrial adaptor TRAK2 activates and functionally links opposing kinesin and dynein motors . Nat Commun 12 , 4578 . doi: 10.1038/s41467-021-24862-7 . OpenUrl CrossRef PubMed 30. MacAskill , A.F. , Brickley , K. , Stephenson , F.A. , and Kittler , J.T . ( 2009 ). GTPase dependent recruitment of Grif-1 by Miro1 regulates mitochondrial trafficking in hippocampal neurons . Mol Cell Neurosci 40 , 301 – 312 . doi: 10.1016/j.mcn.2008.10.016 . OpenUrl CrossRef PubMed Web of Science 31. ↵ MacAskill , A.F. , and Kittler , J.T . ( 2010 ). Control of mitochondrial transport and localization in neurons . Trends Cell Biol 20 , 102 – 112 . doi: 10.1016/j.tcb.2009.11.002 . OpenUrl CrossRef PubMed Web of Science 32. ↵ Han , S.M. , Baig , H.S. , and Hammarlund , M . ( 2016 ). Mitochondria Localize to Injured Axons to Support Regeneration . Neuron 92 , 1308 – 1323 . doi: 10.1016/j.neuron.2016.11.025 . OpenUrl CrossRef PubMed 33. Zhou , B. , Yu , P. , Lin , M.Y. , Sun , T. , Chen , Y. , and Sheng , Z.H . ( 2016 ). Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits . J Cell Biol 214 , 103 – 119 . doi: 10.1083/jcb.201605101 . OpenUrl Abstract / FREE Full Text 34. ↵ Campbell , P.D. , Shen , K. , Sapio , M.R. , Glenn , T.D. , Talbot , W.S. , and Marlow , F.L . ( 2014 ). Unique function of Kinesin Kif5A in localization of mitochondria in axons . J Neurosci 34 , 14717 – 14732 . doi: 10.1523/JNEUROSCI.2770-14.2014 . OpenUrl Abstract / FREE Full Text 35. ↵ Drerup , C.M. , Herbert , A.L. , Monk , K.R. , and Nechiporuk , A.V . ( 2017 ). Regulation of mitochondria-dynactin interaction and mitochondrial retr ograde transport in axons . eLife 6 , e22234 . doi: 10.7554/eLife.22234 . OpenUrl CrossRef 36. Joshi , D.C. , Zhang , C.L. , Babujee , L. , Vevea , J.D. , August , B.K. , Sheng , Z.H. , Chapman , E.R. , Gomez , T.M. , and Chiu , S.Y . ( 2019 ). Inappropriate Intrusion of an Axonal Mitochondrial Anchor into Dendrites Causes Neurodegeneration . Cell Rep 29 , 685 – 696 e685. doi: 10.1016/j.celrep.2019.09.012 . OpenUrl CrossRef PubMed 37. ↵ Mandal , A. , Wong , H.-T.C. , Pinter , K. , Mosqueda , N. , Beirl , A. , Lomash , R.M. , Won , S. , Kindt , K.S. , and Drerup , C.M . ( 2021 ). Retrograde Mitochondrial Transport Is Essential for Organelle Distribution and Health in Zebrafish Neurons . The Journal of Neuroscience 41 , 1371 – 1392 . doi: 10.1523/JNEUROSCI.1316-20.2020 . OpenUrl Abstract / FREE Full Text 38. ↵ Metcalfe , W.K. , Kimmel , C.B. , and Schabtach , E . ( 1985 ). Anatomy of the posterior lateral line system in young larvae of the zebrafish . The Journal of Comparative Neurology 233 , 377 – 389 . doi: 10.1002/cne.902330307 . OpenUrl CrossRef PubMed Web of Science 39. ↵ Kindt , Katie S. , Finch , G. , and Nicolson , T . ( 2012 ). Kinocilia Mediate Mechanosensitivity in Developing Zebrafish Hair Cells . Developmental Cell 23 , 329 – 341 . doi: 10.1016/j.devcel.2012.05.022 . OpenUrl CrossRef PubMed Web of Science 40. ↵ Eckley , D.M. , and Schroer , T.A . ( 2003 ). Interactions between the evolutionarily conserved, actin-related protein, Arp11, actin, and Arp1 . Mol Biol Cell 14 , 2645 – 2654 . doi: 10.1091/mbc.e03-01-0049 . OpenUrl Abstract / FREE Full Text 41. ↵ Choi , H.M.T. , Schwarzkopf , M. , Fornace , M.E. , Acharya , A. , Artavanis , G. , Stegmaier , J. , Cunha , A. , and Pierce , N.A . ( 2018 ). Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust . Development 145 , dev165753. doi: 10.1242/dev.165753 . OpenUrl Abstract / FREE Full Text 42. ↵ Kawano , D. , Pinter , K. , Chlebowski , M. , Petralia , R.S. , Wang , Y.-X. , Nechiporuk , A.V. , and Drerup , C.M . ( 2022 ). NudC regulated Lis1 stability is essential for the maintenance of dynamic microtubule ends in axon terminals . iScience 25 , 105072 . doi: 10.1016/j.isci.2022.105072 . OpenUrl CrossRef PubMed 43. ↵ Lloyd , Thomas E. , Machamer , J. , O’Hara , K. , Kim , Ji H. , Collins , Sarah E. , Wong , Man Y. , Sahin , B. , Imlach , W. , Yang , Y. , Levitan , Edwin S. , et al. ( 2012 ). The p150Glued CAP-Gly Domain Regulates Initiation of Retrograde Transp ort at Synaptic Termini . Neuron 74 , 344 – 360 . doi: 10.1016/j.neuron.2012.02.026 . OpenUrl CrossRef PubMed 44. McGrail , M. , Gepner , J. , Silvanovich , A. , Ludmann , S. , Serr , M. , and Hays , T.S . ( 1995 ). Regulation of cytoplasmic dynein function in vivo by the Drosophila Glued complex . The Journal of cell biology 131 , 411 – 425 . doi: 10.1083/jcb.131.2.411 . OpenUrl Abstract / FREE Full Text 45. ↵ Moughamian , Armen J. , and Holzbaur , Erika L.F . ( 2012 ). Dynactin Is Required for Transport Initiation from the Distal Axon . Neuron 74 , 331 – 343 . doi: 10.1016/j.neuron.2012.02.025 . OpenUrl CrossRef PubMed Web of Science 46. ↵ Young , M.J. , Humble , M.M. , DeBalsi , K.L. , Sun , K.Y. , and Copeland , W.C . ( 2015 ). POLG2 disease variants: analyses reveal a dominant negative heterodimer, altered mitochondrial localization and impaired respiratory capacity . Hum Mol Genet 24 , 5184 – 5197 . doi: 10.1093/hmg/ddv240 . OpenUrl CrossRef PubMed 47. ↵ Rajala , N. , Gerhold , J.M. , Martinsson , P. , Klymov , A. , and Spelbrink , J.N . ( 2014 ). Replication factors transiently associate with mtDNA at the mitochondrial inner membrane to facilitate replication . Nucleic Acids Research 42 , 952 – 967 . doi: 10.1093/nar/gkt988 . OpenUrl CrossRef PubMed 48. ↵ Kaufman , B.A. , Durisic , N. , Mativetsky , J.M. , Costantino , S. , Hancock , M.A. , Grutter , P. , and Shoubridge , E.A . ( 2007 ). The Mitochondrial Transcription Factor TFAM Coordinates the Assembly of Multiple DNA Molecules into Nucleoid-like Structures . Molecular Biology of the Cell 18 , 3225 – 3236 . doi: 10.1091/mbc.e07-05-0404 . OpenUrl Abstract / FREE Full Text 49. ↵ Rath , S. , Sharma , R. , Gupta , R. , Ast , T. , Chan , C. , Durham , T.J. , Goodman , R.P. , Grabarek , Z. , Haas , M.E. , Hung , W.H.W. , et al. ( 2020 ). MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations . Nucleic Acids Research 49 , D1541 – D1547 . doi: 10.1093/nar/gkaa1011 . OpenUrl CrossRef PubMed 50. ↵ Ghysen , A. , and Dambly-Chaudiere , C . ( 2005 ). The three-sided romance of the lateral line: glia love axons love precursors love glia . Bioessays 27 , 488 – 494 . doi: 10.1002/bies.20225 . OpenUrl CrossRef PubMed 51. ↵ Wong , H.-T.C. , Lang , A.E. , Stein , C. , and Drerup , C.M . ( 2024 ). ALS-Linked VapB P56S Mutation Alters Neuronal Mitochondrial Turnover at the Synapse . The Journal of Neuroscience 44 , e0879242024 . doi: 10.1523/JNEUROSCI.0879-24.2024 . OpenUrl Abstract / FREE Full Text 52. ↵ Mandal , A. , Pinter , K. , and Drerup , C.M . ( 2018 ). Analyzing Neuronal Mitochondria in vivo Using Fluorescent Reporters in Zebrafish . Frontiers in Cell and Developmental Biology 6 , 144 . doi: 10.3389/fcell.2018.00144 . OpenUrl CrossRef PubMed 53. ↵ Kageyama , Y. , Zhang , Z. , Roda , R. , Fukaya , M. , Wakabayashi , J. , Wakabayashi , N. , Kensler , T.W. , Reddy , P.H. , Iijima , M. , and Sesaki , H . ( 2012 ). Mitochondrial division ensures the survival of postmitotic neurons by suppressing oxidative damage . J Cell Biol 197 , 535 – 551 . doi: 10.1083/jcb.201110034 . OpenUrl Abstract / FREE Full Text 54. ↵ Smirnova , E. , Griparic , L. , Shurland , D.L. , and van der Bliek , A.M . ( 2001 ). Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells . Mol Biol Cell 12 , 2245 – 2256 . doi: 10.1091/mbc.12.8.2245 . OpenUrl Abstract / FREE Full Text 55. ↵ Zhu , P.P. , Patterson , A. , Stadler , J. , Seeburg , D.P. , Sheng , M. , and Blackstone , C . ( 2004 ). Intra- and intermolecular domain interactions of the C-terminal GTPase effector domain of the multimeric dynamin-like GTPase Drp1 . J Biol Chem 279 , 35967 – 35974 . doi: 10.1074/jbc.M404105200 . OpenUrl Abstract / FREE Full Text 56. ↵ Renken , C.J. , Kim , S. , Wu , Y. , Hammarlund , M. , and Yogev , S . ( 2024 ). Cytoplasmic ribosomes hitchhike on mitochondria to dendrites . doi: 10.1101/2024.09.13.612863 . OpenUrl Abstract / FREE Full Text 57. ↵ Horie , C. , Suzuki , H. , Sakaguchi , M. , and Mihara , K . ( 2002 ). Characterization of Signal That Directs C-Tail–anchored Proteins to Mammalian Mitochondrial Outer Membrane . Molecular Biology of the Cell 13 , 1615 – 1625 . doi: 10.1091/mbc.01-12-0570 . OpenUrl Abstract / FREE Full Text 58. ↵ Yang , R. , Bentley , M. , Huang , C.-F. , and Banker , G . ( 2016 ). Analyzing kinesin motor domain translocation in cultured hippocampal neurons. In Methods in Cell Biology , (Elsevier ), pp. 217 – 232 . 59. ↵ Eichner , L.J. , and Giguère , V . ( 2011 ). Estrogen related receptors (ERRs): A new dawn in transcriptional control of mitochondrial gene networks . Mitochondrion 11 , 544 – 552 . doi: 10.1016/j.mito.2011.03.121 . OpenUrl CrossRef PubMed Web of Science 60. ↵ Hock , M.B. , and Kralli , A . ( 2009 ). Transcriptional Control of Mitochondrial Biogenesis and Function . Annual Review of Physiology 71 , 177 – 203 . doi: 10.1146/annurev.physiol.010908.163119 . OpenUrl CrossRef PubMed Web of Science 61. ↵ Fan , W. , He , N. , Lin , C.S. , Wei , Z. , Hah , N. , Waizenegger , W. , He , M.-X. , Liddle , C. , Yu , R.T. , Atkins , A.R. , et al. ( 2018 ). ERRγ Promotes Angiogenesis, Mitochondrial Biogenesis, and Oxidative Remodeling in PGC1α/β-Deficient Muscle . Cell Reports 22 , 2521 – 2529 . doi: 10.1016/j.celrep.2018.02.047 . OpenUrl CrossRef PubMed 62. ↵ Huss , J.M. , Torra , I.P. , Staels , B. , Giguère , V. , and Kelly , D.P . ( 2004 ). Estrogen-Related Receptor α Directs Peroxisome Proliferator-Activated Receptor α Signaling in the Transcriptional Control of Energy Metabolism in Cardiac and Skeletal Muscle . Molecular and Cellular Biology 24 , 9079 – 9091 . doi: 10.1128/MCB.24.20.9079-9091.2004 . OpenUrl Abstract / FREE Full Text 63. ↵ Pei , L. , Mu , Y. , Leblanc , M. , Alaynick , W. , Barish , Grant D. , Pankratz , M. , Tseng , Tiffany W. , Kaufman , S. , Liddle , C. , Yu , Ruth T. , et al. ( 2015 ). Dependence of Hippocampal Function on ERRγ-Regulated Mitochondrial Metabolism . Cell Metabolism 21 , 628 – 636 . doi: 10.1016/j.cmet.2015.03.004 . OpenUrl CrossRef PubMed 64. ↵ Howitz , K.T. , Bitterman , K.J. , Cohen , H.Y. , Lamming , D.W. , Lavu , S. , Wood , J.G. , Zipkin , R.E. , Chung , P. , Kisielewski , A. , Zhang , L.-L. , et al. ( 2003 ). Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan . Nature 425 , 191 – 196 . doi: 10.1038/nature01960 . OpenUrl CrossRef PubMed Web of Science 65. ↵ Lopes Costa , A. , Le Bachelier , C. , Mathieu , L. , Rotig , A. , Boneh , A. , De Lonlay , P. , Tarnopolsky , M.A. , Thorburn , D.R. , Bastin , J. , and Djouadi , F . ( 2014 ). Beneficial effects of resveratrol on respiratory chain defects in patients’ fibroblasts involve estrogen receptor and estrogen-related receptor alpha signaling . Human Molecular Genetics 23 , 2106 – 2119 . doi: 10.1093/hmg/ddt603 . OpenUrl CrossRef PubMed 66. Sopariwala , D.H. , Rios , A.S. , Park , M.K. , Song , M.S. , Kumar , A. , and Narkar , V.A . ( 2022 ). Estrogen-related receptor alpha is an AMPK -regulated factor that promotes ischemic muscle revascularization and recovery in diet-induced obese mice . FASEB BioAdvances 4 , 602 – 618 . doi: 10.1096/fba.2022-00015 . OpenUrl CrossRef PubMed 67. ↵ Wilson , B.J. , Tremblay , A.M. , Deblois , G. , Sylvain-Drolet , G. , and Giguère , V . ( 2010 ). An Acetylation Switch Modulates the Transcriptional Activity of Estrogen-Related Receptor α . Molecular Endocrinology 24 , 1349 – 1358 . doi: 10.1210/me.2009-0441 . OpenUrl CrossRef PubMed Web of Science 68. ↵ Yan , M. , Audet-Walsh , É. , Manteghi , S. , Dufour , C.R. , Walker , B. , Baba , M. , St-Pierre , J. , Giguère , V. , and Pause , A . ( 2016 ). Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1α/ERRα . Genes & Development 30 , 1034 – 1046 . doi: 10.1101/gad.281410.116 . OpenUrl Abstract / FREE Full Text 69. ↵ Corton , J.M. , Gillespie , J.G. , Hawley , S.A. , and Hardie , D.G . ( 1995 ). 5-Aminoimidazole-4-Carboxamide Ribonucleoside. A Specific Method for Activating AMP-Activated Protein Kinase in Intact Cells? European Journal of Biochemistry 229 , 558 – 565 . doi: 10.1111/j.1432-1033.1995.tb20498.x . OpenUrl CrossRef PubMed Web of Science 70. ↵ Mosser , E.A. , Chiu , C.N. , Tamai , T.K. , Hirota , T. , Li , S. , Hui , M. , Wang , A. , Singh , C. , Giovanni , A. , Kay , S.A. , and Prober , D.A . ( 2019 ). Identification of pathways that regulate circadian rhythms using a larval zebrafish small molecule screen . Scientific Reports 9 , 12405 . doi: 10.1038/s41598-019-48914-7 . OpenUrl CrossRef PubMed 71. ↵ Johnston , S.D. , Liu , X. , Zuo , F. , Eisenbraun , T.L. , Wiley , S.R. , Kraus , R.J. , and Mertz , J.E . ( 1997 ). Estrogen-Related Receptor α1 Functionally Binds as a Monomer to Extended Half-Site Sequences Including Ones Contained within Estrogen-Response Elements . Molecular Endocrinology 11 , 342 – 352 . doi: 10.1210/mend.11.3.9897 . OpenUrl CrossRef PubMed Web of Science 72. ↵ Blander , G. , and Guarente , L . ( 2004 ). The Sir2 family of protein deacetylases . Annu Rev Biochem 73 , 417 – 435 . doi: 10.1146/annurev.biochem.73.011303.073651 . OpenUrl CrossRef PubMed Web of Science 73. ↵ McMeekin , L.J. , Joyce , K.L. , Jenkins , L.M. , Bohannon , B.M. , Patel , K.D. , Bohannon , A.S. , Patel , A. , Fox , S.N. , Simmons , M.S. , Day , J.J. , et al. ( 2021 ). Estrogen-related Receptor Alpha (ERRα) is Required for PGC-1α-dependent Gene Expression in the Mouse Brain . Neuroscience 479 , 70 – 90 . doi: 10.1016/j.neuroscience.2021.10.007 . OpenUrl CrossRef 74. ↵ Alano , C.C. , Tran , A. , Tao , R. , Ying , W. , Karliner , J.S. , and Swanson , R.A . ( 2007 ). Differences among cell types in NAD(+) compartmentalization: a comparison of neurons, astrocytes, and cardiac myocytes . J Neurosci Res 85 , 3378 – 3385 . doi: 10.1002/jnr.21479 . OpenUrl CrossRef PubMed Web of Science 75. Hu , Q. , Wu , D. , Walker , M. , Wang , P. , Tian , R. , and Wang , W . ( 2021 ). Genetically encoded biosensors for evaluating NAD(+)/NADH ratio in cytosolic and mitochondrial compartments . Cell Rep Methods 1 . doi: 10.1016/j.crmeth.2021.100116 . OpenUrl CrossRef 76. ↵ Wareski , P. , Vaarmann , A. , Choubey , V. , Safiulina , D. , Liiv , J. , Kuum , M. , and Kaasik , A . ( 2009 ). PGC-1α and PGC-1Β Regulate Mitochondrial Density in Neurons . Journal of Biological Chemistry 284 , 21379 – 21385 . doi: 10.1074/jbc.M109.018911 . OpenUrl Abstract / FREE Full Text 77. ↵ Chen , Y. , Jiang , Y. , Yang , Y. , Huang , X. , and Sun , C . ( 2021 ). SIRT1 Protects Dopaminergic Neurons in Parkinson’s Disease Models via PGC-1α-Mediated Mitochondrial Biogenesis . Neurotoxicity Research 39 , 1393 – 1404 . doi: 10.1007/s12640-021-00392-4 . OpenUrl CrossRef PubMed 78. ↵ Hatsuda , A. , Kurisu , J. , Fujishima , K. , Kawaguchi , A. , Ohno , N. , and Kengaku , M . ( 2023 ). Calcium signals tune AMPK activity and mitochondrial homeostasis in dendrites of developing neurons . Development 150 , dev201930. doi: 10.1242/dev.201930 . OpenUrl CrossRef PubMed 79. ↵ Eudy , J.D. , Yao , S. , Weston , M.D. , Ma-Edmonds , M. , Talmadge , C.B. , Cheng , J.J. , Kimberling , W.J. , and Sumegi , J . ( 1998 ). Isolation of a Gene Encoding a Novel Member of the Nuclear Receptor Superfamily from the Critical Region of Usher Syndrome Type IIa at 1q41 . Genomics 50 , 382 – 384 . doi: 10.1006/geno.1998.5345 . OpenUrl CrossRef PubMed Web of Science 80. ↵ Giguère , V. , Yang , N. , Segui , P. , and Evans , R.M . ( 1988 ). Identification of a new class of steroid hormone receptors . Nature 331 , 91 – 94 . doi: 10.1038/331091a0 . OpenUrl CrossRef PubMed Web of Science 81. ↵ Huss , J.M. , Garbacz , W.G. , and Xie , W . ( 2015 ). Constitutive activities of estrogen-related receptors: Transcriptional regulation of metabolism by the ERR pathways in health and disease . Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1852 , 1912 – 1927 . doi: 10.1016/j.bbadis.2015.06.016 . OpenUrl CrossRef PubMed 82. ↵ Lydon , J.P. , Power , R.F. , and Conneely , O.M . ( 1992 ). Differential modes of activation define orphan subclasses within the steroid/thyroid receptor superfamily . Gene Expr 2 , 273 – 283 . OpenUrl PubMed 83. ↵ Vernier , M. , and Giguère , V . ( 2021 ). Aging, senescence and mitochondria: the PGC-1/ERR axis . Journal of Molecular Endocrinology 66 , R1 – R14 . doi: 10.1530/JME-20-0196 . OpenUrl CrossRef PubMed 84. ↵ Yu , L. , and Yang , S.J . ( 2010 ). AMP-activated protein kinase mediates activity-dependent regulation of peroxisome proliferator-activated receptor γ coactivator-1α and nuclear respiratory factor 1 expression in rat visual cortical neurons . Neuroscience 169 , 23 – 38 . doi: 10.1016/j.neuroscience.2010.04.063 . OpenUrl CrossRef PubMed Web of Science 85. ↵ Lagouge , M. , Argmann , C. , Gerhart-Hines , Z. , Meziane , H. , Lerin , C. , Daussin , F. , Messadeq , N. , Milne , J. , Lambert , P. , Elliott , P. , et al. ( 2006 ). Resveratrol Improves Mitochondrial Function and Protects against Metabolic Disease by Activating SIRT1 and PGC-1α . Cell 127 , 1109 – 1122 . doi: 10.1016/j.cell.2006.11.013 . OpenUrl CrossRef PubMed Web of Science 86. ↵ Michishita , E. , Park , J.Y. , Burneskis , J.M. , Barrett , J.C. , and Horikawa , I . ( 2005 ). Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins . Mol Biol Cell 16 , 4623 – 4635 . doi: 10.1091/mbc.e05-01-0033 . OpenUrl Abstract / FREE Full Text 87. ↵ Qin , W. , Haroutunian , V. , Katsel , P. , Cardozo , C.P. , Ho , L. , Buxbaum , J.D. , and Pasinetti , G.M . ( 2009 ). PGC-1α Expression Decreases in the Alzheimer Disease Brain as a Function of Dementia . Archives of Neurology 66 . doi: 10.1001/archneurol.2008.588 . OpenUrl CrossRef PubMed Web of Science 88. Sheng , B. , Wang , X. , Su , B. , Lee , H.-g ., Casadesus , G ., Perry , G. , and Zhu , X . ( 2012 ). Impaired mitochondrial biogenesis contributes to mitochondrial dysfunction in Alzheimer’s disease: Impaired mitochondrial biogenesis in AD . Journal of Neurochemistry 120 , 419 – 429 . doi: 10.1111/j.1471-4159.2011.07581.x . OpenUrl CrossRef PubMed Web of Science 89. Shin , J.-H. , Ko , Han S. , Kang , H. , Lee , Y. , Lee , Y.-I. , Pletinkova , O. , Troconso , Juan C. , Dawson , Valina L. , and Dawson , Ted M . ( 2011 ). PARIS (ZNF746) Repression of PGC-1α Contributes to Neurodegeneration in Parkinson’s Disease . Cell 144 , 689 – 702 . doi: 10.1016/j.cell.2011.02.010 . OpenUrl CrossRef PubMed Web of Science 90. ↵ Stevens , D.A. , Lee , Y. , Kang , H.C. , Lee , B.D. , Lee , Y.-I. , Bower , A. , Jiang , H. , Kang , S.-U. , Andrabi , S.A. , Dawson , V.L. , et al. ( 2015 ). Parkin loss leads to PARIS-dependent declines in mitochondrial mass and respiration . Proceedings of the National Academy of Sciences 112 , 11696 – 11701 . doi: 10.1073/pnas.1500624112 . OpenUrl Abstract / FREE Full Text 91. ↵ Westerfield , M . ( 1993 ). The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio) . 92. ↵ Kimmel , C.B. , Ballard , W.W. , Kimmel , S.R. , Ullmann , B. , and Schilling , T.F . ( 1995 ). Stages of embryonic development of the zebrafish . Developmental Dynamics 203 , 253 – 310 . doi: 10.1002/aja.1002030302 . OpenUrl CrossRef PubMed Web of Science 93. ↵ Kwan , K.M. , Fujimoto , E. , Grabher , C. , Mangum , B.D. , Hardy , M.E. , Campbell , D.S. , Parant , J.M. , Yost , H.J. , Kanki , J.P. , and Chien , C.B . ( 2007 ). The Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructs . Developmental Dynamics 236 , 3088 – 3099 . doi: 10.1002/dvdy.21343 . OpenUrl CrossRef PubMed Web of Science 94. ↵ Gibson , D.G. , Young , L. , Chuang , R.-Y. , Venter , J.C. , Hutchison , C.A. , and Smith , H.O . ( 2009 ). Enzymatic assembly of DNA molecules up to several hundred kilobases . Nature Methods 6 , 343 – 345 . doi: 10.1038/nmeth.1318 . OpenUrl CrossRef PubMed Web of Science 95. ↵ Huang , C.f ., and Banker , G . ( 2012 ). The Translocation Selectivity of the Kinesins that Mediate Neuronal Organelle Transport . Traffic 13 , 549 – 564 . doi: 10.1111/j.1600-0854.2011.01325.x . OpenUrl CrossRef PubMed Web of Science 96. ↵ Cambronne , X.A. , Stewart , M.L. , Kim , D. , Jones-Brunette , A.M. , Morgan , R.K. , Farrens , D.L. , Cohen , M.S. , and Goodman , R.H . ( 2016 ). Biosensor reveals multiple sources for mitochondrial NAD(+) . Science 352 , 1474 – 1477 . doi: 10.1126/science.aad5168 . OpenUrl Abstract / FREE Full Text 97. ↵ Zhang , Q. , Li , S. , Wong , H.C. , He , X.J. , Beirl , A. , Petralia , R.S. , Wang , Y.X. , and Kindt , K.S . ( 2018 ). Synaptically silent sensory hair cells in zebrafish are recruited after damage . Nat Commun 9 , 1388 . doi: 10.1038/s41467-018-03806-8 . OpenUrl CrossRef PubMed 98. ↵ Mo , W. , and Nicolson , T . ( 2011 ). Both Pre- and Postsynaptic Activity of Nsf Prevents Degeneration of Hair-Cell Synapses . PLoS ONE 6 , e27146 . doi: 10.1371/journal.pone.0027146 . OpenUrl CrossRef PubMed 99. ↵ Drerup , C.M. , and Nechiporuk , A.V . ( 2013 ). JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes . PLoS Genetics 9 , e1003303 . doi: 10.1371/journal.pgen.1003303 . OpenUrl CrossRef PubMed 100. ↵ Drerup , C.M. , and Nechiporuk , A.V . ( 2016 ). In vivo analysis of axonal transport in zebrafish . Methods in cell biology 131 , 311 – 329 . doi: 10.1016/bs.mcb.2015.06.007 . OpenUrl CrossRef PubMed 101. ↵ Preibisch , S. , Saalfeld , S. , and Tomancak , P . ( 2009 ). Globally optimal stitching of tiled 3D microscopic image acquisitions . Bioinformatics 25 , 1463 – 1465 . doi: 10.1093/bioinformatics/btp184 . OpenUrl CrossRef PubMed Web of Science 102. ↵ Schindelin , J. , Arganda-Carreras , I. , Frise , E. , Kaynig , V. , Longair , M. , Pietzsch , T. , Preibisch , S. , Rueden , C. , Saalfeld , S. , Schmid , B ., et al. ( 2012 ). Fiji : an open-source platform for biological-image analysis . Nature Methods 9 , 676 – 682 . doi: 10.1038/nmeth.2019 . OpenUrl CrossRef PubMed Web of Science 103. ↵ Thisse , C. , and Thisse , B . ( 2008 ). High-resolution in situ hybridization to whole-mount zebrafish embryos . Nature Protocols 3 , 59 – 69 . doi: 10.1038/nprot.2007.514 . OpenUrl CrossRef PubMed Web of Science 104. ↵ Logel , J. , Dill , D. , and Leonard , S . ( 1992 ). Synthesis of cRNA probes from PCR-generated DNA . BioTechniques 13 , 604 – 610 . OpenUrl PubMed Web of Science 105. ↵ Bolger , A.M. , Lohse , M. , and Usadel , B . ( 2014 ). Trimmomatic: a flexible trimmer for Illumina sequence data . Bioinformatics 30 , 2114 – 2120 . doi: 10.1093/bioinformatics/btu170 . OpenUrl CrossRef PubMed Web of Science 106. ↵ Dobin , A. , Davis , C.A. , Schlesinger , F. , Drenkow , J. , Zaleski , C. , Jha , S. , Batut , P. , Chaisson , M. , and Gingeras , T.R . ( 2013 ). STAR: ultrafast universal RNA-seq aligner . Bioinformatics 29 , 15 – 21 . doi: 10.1093/bioinformatics/bts635 . OpenUrl CrossRef PubMed Web of Science 107. ↵ Liao , Y. , Smyth , G.K. , and Shi , W . ( 2014 ). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features . Bioinformatics 30 , 923 – 930 . doi: 10.1093/bioinformatics/btt656 . OpenUrl CrossRef PubMed Web of Science 108. ↵ Love , M.I. , Huber , W. , and Anders , S . ( 2014 ). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 . Genome Biology 15 , 550 . doi: 10.1186/s13059-014-0550-8 . OpenUrl CrossRef PubMed 109. ↵ Yu , G . ( 2024 ). Thirteen years of clusterProfiler . The Innovation 5 , 100722 . doi: 10.1016/j.xinn.2024.100722 . OpenUrl CrossRef PubMed 110. ↵ Kolberg , L. , Raudvere , U. , Kuzmin , I. , Adler , P. , Vilo , J. , and Peterson , H . ( 2023 ). g:Profiler—interoperable web service for functional enrichment analysis and gene identifier mapping (2023 update) . Nucleic Acids Research 51 , W207 – W212 . doi: 10.1093/nar/gkad347 . OpenUrl CrossRef 111. ↵ Puente-Santamaria , L. , Wasserman , W.W. , and Del Peso , L . ( 2019 ). TFEA.ChIP: a tool kit for transcription factor binding site enrichment analysis capitalizing on ChIP-seq datasets . Bioinformatics 35 , 5339 – 5340 . doi: 10.1093/bioinformatics/btz573 . OpenUrl CrossRef PubMed 112. ↵ Elizarraras , J.M. , Liao , Y. , Shi , Z. , Zhu , Q. , Pico , Alexander R. , and Zhang , B . ( 2024 ). WebGestalt 2024: faster gene set analysis and new support for metabolomics and multi-omics . Nucleic Acids Research 52 , W415 – W421 . doi: 10.1093/nar/gkae456 . OpenUrl CrossRef PubMed 113. ↵ Wickham , H . ( 2016 ). ggplot2: Elegant Graphics for Data Analysis, 2nd 2016 Edition ( Springer International Publishing : Imprint: Springer ). 114. Lister , J.A. , Robertson , C.P. , Lepage , T. , Johnson , S.L. , and Raible , D.W . ( 1999 ). nacre encodes a zebrafish microphthalmia-related protein that regulates neural-crest-derived pigment cell fate . Development 126 , 3757 – 3767 . doi: 10.1242/dev.126.17.3757 . OpenUrl Abstract 115. Obholzer , N. , Wolfson , S. , Trapani , J.G. , Mo , W. , Nechiporuk , A. , Busch-Nentwich , E. , Seiler , C. , Sidi , S. , Söllner , C. , Duncan , R.N. , et al. ( 2008 ). Vesicular Glutamate Transporter 3 Is Required for Synaptic Transmission in Zebrafish Hair Cells . The Journal of Neuroscience 28 , 2110 – 2118 . doi: 10.1523/JNEUROSCI.5230-07.2008 . OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted April 15, 2026. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Retrograde mitochondrial transport is required for mitochondrial biogenesis in zebrafish neurons Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Retrograde mitochondrial transport is required for mitochondrial biogenesis in zebrafish neurons Angelica E Lang , Chris Stein , Roger Schultz , Catherine M Drerup bioRxiv 2025.09.29.679307; doi: https://doi.org/10.1101/2025.09.29.679307 Share This Article: Copy Citation Tools Retrograde mitochondrial transport is required for mitochondrial biogenesis in zebrafish neurons Angelica E Lang , Chris Stein , Roger Schultz , Catherine M Drerup bioRxiv 2025.09.29.679307; doi: https://doi.org/10.1101/2025.09.29.679307 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7618) Biochemistry (17633) Bioengineering (13857) Bioinformatics (41841) Biophysics (21399) Cancer Biology (18529) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24282) Genetics (15582) Genomics (22462) Immunology (17700) Microbiology (40295) Molecular Biology (17140) Neuroscience (88421) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4813) Physiology (7632) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4284) Systems Biology (9808) Zoology (2267)