miR126-5p Downregulation Facilitates Axon Degeneration and NMJ Disruption via a Non-Cell-Autonomous Mechanism in ALS.

Axon degeneration and disruption of neuromuscular junctions (NMJs) are key events in amyotrophic lateral sclerosis (ALS) pathology. Although the disease\u27s etiology is not fully understood, it is thought to involve a non-cell-autonomous mechanism and alterations in RNA metabolism. Here, we identified reduced levels of miR126-5p in presymptomatic ALS male mice models, and an increase in its targets: axon destabilizing Type 3 Semaphorins and their coreceptor Neuropilins. Using compartmentalize

Elimination of aberrant DRG circuitries in Sema3A mutant mice leads to extensive neuronal deficits.

Axon guidance molecules determine the pattern of neuronal circuits. Accuracy of the process is ensured by unknown mechanisms that correct early guidance errors. Since the time frame of error correction in Sema3A null mice partly overlaps with the period of naturally occurring cell death in dorsal root ganglia (DRG) development, we tested the hypothesis that apoptosis of misguided neurons enables error correction. We crossed BAX null mice, in which DRG apoptosis is blocked, with Sema3A null mice to induce errors. Analyses of these double-null mouse embryos showed that the elimination of abnormal projections is not blocked in the absence of BAX. Surprisingly however, there are fewer surviving neurons in Sema3A null or Sema3A/BAX double-null newborn mice than in wild-type mice. These results suggest that guidance errors are corrected by a BAX-independent cell death mechanism. Thus, aberrant axonal guidance may lead to reductions in neuronal numbers to suboptimal levels, perhaps increasing the likelihood of neuropathological consequences later in life

Increased levels of axon guidance errors in BAX null and heterozygote Sema3A:BAX null mice.

<p><b>A:</b> Representative image of errors observed in DRG from Sema3A^+/+:BAX^−/− mice. Three adjacent cross sections of E13.5 DRG are shown. Most of the observed errors in the indicated genotypes were single axons or small bundles. Note the tortuous path an abnormal axon takes in and out of the plane of view. Scale bar 50 µm. <b>B:</b> Analysis of error occurrence in the indicated genotypes at E13.5. Increase in error frequency is detected in BAX null background, indicating cell-death involvement in both wild-type and Sem3A^+/− mice (n = 30 DRG per genotype (Wild type versus Sema3A^+/+:BAX^−/− P = 0.0416, Sema3A^+/− :BAX^+/+ versus Sema3A^+/−:BAX^−/− P = 0.0006). <b>C:</b> DRG neurons from BAX^−/− and BAX^+/+ mice are equally responsive to Sema3A-induced growth cone collapse. DRG explants from E12.5 embryos (BAX^+/+ and BAX^−/− littermates) were cultured in the presence of 10 ng/ml NGF for 20 h, at which time neurons were treated with 0, 6, 7.5, 10, 15 or 30 pM Sema3A. After an additional incubation period of 40 min with or without Sema3A, the explants were fixed and stained with rhodamine phalloidin. Quantification of the growth cone collapse results is shown. Results represent the mean +/− S.E.M. of three independent experiments. None of the Sema3A concentrations induced statistically significant difference in collapse levels between BAX^+/+ and BAX^−/− groups, P>0.05. <b>D:</b> Activation of caspase-3 is not significantly changed in Sema3A null mice. For each embryo proteins were extracted from E15.5 (upper panel) and E16.5 (lower panel) DRGs from lumbar and thoracic levels (at least three embryos of each genotype were used). Relative changes in caspase-3 activation levels were measured by western blot analysis using an activated caspase-3-specific antibody (Cell Signaling Technology). To determine protein levels each membrane was re-blotted for actin. Quantification of band intensity was obtained using scanning densitometry (Quantity One, BioRad) of three blots representing three different experiments. Results were normalized to actin. The average normalized result of the wild-type embryos at each age was defined as 1. At E15.5 the difference between Sema3A null mice and wild-type littermates is not statistically significant (P>0.05).</p

Abnormal axon projection elimination is not blocked in the absence of BAX.

<p><b>A:</b> Illustrated cross section of the lumbar spinal cord at E15.5 used to analyze abnormal axon projections. Spinal cord in the middle (red circle) with DRG on both sides (red star) is surrounded by pre-cartilage primordial (red rhombus). The left side portrays an abnormal circuit – pseudo-uni-bipolar neurons within the DRG (blue) send branches and enter the spinal cord at the dorsal root entry (green arrow). A second branch crosses abnormally through the pre-cartilage primordial (purple arrow) instead of using the correct path through the ventral root (green arrow – same marking shapes are used hereafter). The right side portrays a normal circuit (note that the ventral root contains the ventral nerve of the DRG and the motor nerve exiting the spinal cord ventrally). <b>B:</b> Experimental design. Mouse genetics used to test the role of apoptosis in the elimination of aberrant circuits. C<b>:</b> Abnormal axon projections in three adjacent cross-sections of DRG from a Sema3A^−/− embryo (E15.5). Note the normal dorsal and ventral roots (short green arrows, upper right image). Abnormal projections were scored only when actual exit from the DRG was noticeable (arrows in upper and lower left images). Serial sections were used to identify <i>bona fide</i> errors. Low magnification in upper panel and high magnification of a different error in lower panel; note that single axon errors could be detected at this magnification (lower right image, purple arrowhead). All scale bars 50 µm. <b>D:</b> Similar axon errors are detected in Sema3A null mice and Sema3A:BAX double null mice. Abnormal axon projections in Sema3A^−/−:BAX^+/+ mice (left) or Sema3A^−/−:BAX^−/− mutant mice (right). The abnormalities are similar in both cases. A representative image of lumbar DRG cross section is shown (low magnification, upper panel) and (higher magnification, lower panel) of the same abnormal projections (anti-neurofilament immunostaining). At E15.5, these DRGs of Sema3A^−/−:BAX^+/+ and Sema3A^−/−: BAX^−/− embryos exhibit abnormal axon bundles (purple arrow). Spinal cord is marked with a red circle, the pre-cartilage primordial marked with a red rhombus, and the DRG is marked with a red star. All scale bars are 50 µm. <b>E:</b> Analysis of temporal error occurrence. Error rate over time in Sema3A^−/: BAX^+/+ embryos compared to error rate in the compound Sema3A^−/−:BAX^−/− embryos. Absence of BAX results in accumulation of errors between E13.5 and E15.5. Massive error elimination is apparent in both genetic backgrounds at E17.5 (n = 30 DRG per genotype in each of the three embryonic ages, E13.5 P = 0.037, E15.5 P = 0.05, and E17.5 P = 0.0042). <b>Fa,b:</b> Representative images of E13.5 lumbar DRG of a Sema3A^−/− embryo cross section are shown (anti-neurofilament immunostaining). a, example of DRG that exhibits three abnormal axon bundles. b, example of DRG that exhibits single axon error. <b>G:</b> Quantification of abnormal axon bundles – Absence of BAX results in accumulation of errors between E13.5 and E15.5. Massive error elimination is apparent in both genetic backgrounds at E17.5 (E13.5 P = 0.0348, E15.5 P = 0.0069, and E17.5 P = 0.0404). <b>H:</b> Quantification of single axon errors – No accumulation of single axon errors is observed between E13.5 and E15.5 in the Sema3A^−/−:BAX^−/− genotype. Single axon errors are fully eliminated by E17.5 in the Sema3A^−/− embryos, while Sema3A^−/−:BAX^−/− embryos still exhibit low but detectable levels of errors (n = 30 DRG per genotype at each of the three embryonic ages, E13.5 P>0.05, E15.5 P>0.05, and E17.5 P<0.05 ).</p

The Beneficial Effect of Mitochondrial Transfer Therapy in 5XFAD Mice via Liver–Serum–Brain Response

We recently reported the benefit of the IV transferring of active exogenous mitochondria in a short-term pharmacological AD (Alzheimer’s disease) model. We have now explored the efficacy of mitochondrial transfer in 5XFAD transgenic mice, aiming to explore the underlying mechanism by which the IV-injected mitochondria affect the diseased brain. Mitochondrial transfer in 5XFAD ameliorated cognitive impairment, amyloid burden, and mitochondrial dysfunction. Exogenously injected mitochondria were detected in the liver but not in the brain. We detected alterations in brain proteome, implicating synapse-related processes, ubiquitination/proteasome-related processes, phagocytosis, and mitochondria-related factors, which may lead to the amelioration of disease. These changes were accompanied by proteome/metabolome alterations in the liver, including pathways of glucose, glutathione, amino acids, biogenic amines, and sphingolipids. Altered liver metabolites were also detected in the serum of the treated mice, particularly metabolites that are known to affect neurodegenerative processes, such as carnosine, putrescine, C24:1-OH sphingomyelin, and amino acids, which serve as neurotransmitters or their precursors. Our results suggest that the beneficial effect of mitochondrial transfer in the 5XFAD mice is mediated by metabolic signaling from the liver via the serum to the brain, where it induces protective effects. The high efficacy of the mitochondrial transfer may offer a novel AD therapy

Alleviation of a polyglucosan storage disorder by enhancement of autophagic glycogen catabolism

Abstract This work employs adult polyglucosan body disease (APBD) models to explore the efficacy and mechanism of action of the polyglucosan‐reducing compound 144DG11. APBD is a glycogen storage disorder (GSD) caused by glycogen branching enzyme (GBE) deficiency causing accumulation of poorly branched glycogen inclusions called polyglucosans. 144DG11 improved survival and motor parameters in a GBE knockin (Gbeys/ys) APBD mouse model. 144DG11 reduced polyglucosan and glycogen in brain, liver, heart, and peripheral nerve. Indirect calorimetry experiments revealed that 144DG11 increases carbohydrate burn at the expense of fat burn, suggesting metabolic mobilization of pathogenic polyglucosan. At the cellular level, 144DG11 increased glycolytic, mitochondrial, and total ATP production. The molecular target of 144DG11 is the lysosomal membrane protein LAMP1, whose interaction with the compound, similar to LAMP1 knockdown, enhanced autolysosomal degradation of glycogen and lysosomal acidification. 144DG11 also enhanced mitochondrial activity and modulated lysosomal features as revealed by bioenergetic, image‐based phenotyping and proteomics analyses. As an effective lysosomal targeting therapy in a GSD model, 144DG11 could be developed into a safe and efficacious glycogen and lysosomal storage disease therapy