Search

The approved gene therapies for spinal muscular atrophy (SMA), caused by loss of survival motor neuron 1 (SMN1), greatly ameliorate SMA natural history but are not curative. These therapies primarily target motor neurons, but SMN1 loss has detrimental effects beyond motor neurons and especially in muscle. Here we show that SMN loss in mouse skeletal muscle leads to accumulation of dysfunctional mitochondria. Expression profiling of single myofibers from a muscle specific Smn1 knockout mouse model revealed down-regulation of mitochondrial and lysosomal genes. Albeit levels of proteins that mark mitochondria for mitophagy were increased, morphologically deranged mitochondria with impaired complex I and IV activity and respiration and that produced excess reactive oxygen species accumulated in Smn1 knockout muscles, because of the lysosomal dysfunction highlighted by the transcriptional profiling. Amniotic fluid stem cells transplantation that corrects the SMN knockout mouse myopathic phenotype restored mitochondrial morphology and expression of mitochondrial genes. Thus, targeting muscle mitochondrial dysfunction in SMA may complement the current gene therapy.

Mitochondria are not only central organelles in metabolism and energy conversion but are also platforms for cellular signaling cascades. Classically, the shape and ultrastructure of mitochondria were depicted as static. The discovery of morphological transitions during cell death and of conserved genes controlling mitochondrial fusion and fission contributed to establishing the concept that mitochondrial morphology and ultrastructure are dynamically regulated by mitochondria-shaping proteins. These finely tuned, dynamic changes in mitochondrial shape can in turn control mitochondrial function, and their alterations in human diseases suggest that this space can be explored for drug discovery. Here, we review the basic tenets and molecular mechanisms of mitochondrial morphology and ultrastructure, describing how they can coordinately define mitochondrial function.

In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5’ AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.

Our group is interested in understanding the mechanisms that contribute to the metabolic and redox coupling between astrocytes and neurons, as well as their implication in cognitive and motor functions. Neurons show a high dependence on mitochondrial oxidative phosphorylation for survival, whereas astrocytes resist to almost complete inhibition of mitochondrial respiration. A key factor in this process is PFKFB3, an enzyme that promotes glycolysis by activating its regulatory enzyme PFK1. We previously demonstrated that PFKFB3 is a substrate of the E3 ubiquitin ligase APC/C-Cdh1. By degrading PFKFB3, APC/C-Cdh1 ac- tivity shifts the equilibrium of glucose consumption from glycolysis towards pentose-phosphate pathway (PPP), thus regulating the redox status and survival of neurons. Conversely, APC/C-Cdh1 activity in astrocytes is very low, which accounts for the elevated PFKFB3 protein levels and glycolytic phenotype of these cells. However, the impact of PFKFB3 modulation in specific brain cells on cognitive and motor functions have not yet been explored. To address this, wgenerated a Cre recombinase inducible ROSA26-floxed knock-in mouse harboring the full-length PFKFB3 cDNA (PFKFB3LoxP/+) to specifically assess the impact of PFKFB3 expression in neurons in vivo. PFKFB3LoxP/+ mice were crossed with mice expressing Cre recombinase under the neuronal CAMKIIa promoter (CAMKIIa-Cre). We found that the resulting PFKFB3LoxP/+; CAMKIIa-Cre mice showed increased PFKFB3 protein levels in neurons in vivo and symptoms of cognitive decline and motor discoordination as from the 3 months of age. Interestingly, this PFKFB3-induced phenotype was rescued by in vivo coexpressing mitoCatalase (mCAT), i.e. the antioxidant enzyme catalase confined to neuronal mitochondria. These results indicate that aberrant enhancement of glycolysis in neurons causes behavioral impairment in mouse through a mitochondrial redox stress-mediated mechanism.

Reactive oxygen species (ROS) are a common product of active mitochondrial respiration carried in mitochondrial cristae, but whether cristae shape influences ROS levels is unclear. Here we report that the mitochondrial fusion and cristae shape protein Opa1 requires mitochondrial ATP synthase oligomers to reduce ROS accumulation. In cells fueled with galactose to force ATP production by mitochondria, cristae are enlarged, ATP synthase oligomers destabilized, and ROS accumulate. Opa1 prevents both cristae remodeling and ROS generation, without impinging on levels of mitochondrial antioxidant defense enzymes that are unaffected by Opa1 overexpression. Genetic and pharmacologic experiments indicate that Opa1 requires ATP synthase oligomerization and activity to reduce ROS levels upon a blockage of the electron transport chain. Our results indicate that the converging effect of Opa1 and mitochondrial ATP synthase on mitochondrial ultrastructure regulate ROS abundance to sustain cell viability.

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in the autophagy-lysosomal pathway causes accumulation of structurally and bioenergetically impaired neuronal mi- tochondria. In vivo genetic approach revealed elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolysis activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFK- FB3, a glycolytic-promoting enzyme normally unstable in healthy neurons. Pharmacological inhibition of PFKFB3 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectified key disease hallmarks. Thus, aberrant upregulation of neuronal glycolysis contributes to CLN7 patho-genesis and targeting PFKFB3 may alleviate this and other lysosomal storage diseases

Metabolism and redox signalling share critical nodes in the nervous system. In the last years, a series of major findings have challenged the current vision on how neural reactive oxygen species (ROS) are produced and handled in the nervous system. Once regarded as deleterious by-products, ROS are now shown to be essential for a metabolic and redox crosstalk. In turn, this coupling defines neural viability and function to control behaviour or leading to neurodegeneration when compromised. Findings like a different assembly of mitochondrial respiratory supercomplexes in neurons and astrocytes stands behind a divergent production of ROS in either cell type, more prominent in astrocytes. ROS levels are however tightly controlled by an antioxidant machinery in astrocytes, assumed as more efficient than that of neurons, to regulate redox signalling. By exerting this control in ROS abundance, metabolic functions are finely tuned in both neural cells. Further, a higher engagement of mitochondrial respiration and oxidative function in neurons, underpinned by redox equivalents supplied from the pentose phosphate pathway and from glia, differs from the otherwise strong glycolytic capacity of astrocytes. Here, we recapitulate major findings on how ROS and metabolism differ between neural cells but merge to define reciprocal signalling pathways, ultimately defining neural function and fate.

Dopamine is involved in physiological processes like learning and memory, motor control and reward, and pathological conditions such as Parkinson's disease and addiction. In contrast to the extensive studies on neurons, astrocyte involvement in dopaminergic signaling remains largely unknown. Using transgenic mice, optogenetics, and pharmacogenetics, we studied the role of astrocytes on the dopaminergic system. We show that in freely behaving mice, astrocytes in the nucleus accumbens (NAc), a key reward center in the brain, respond with Ca elevations to synaptically released dopamine, a phenomenon enhanced by amphetamine. In brain slices, synaptically released dopamine increases astrocyte Ca, stimulates ATP/adenosine release, and depresses excitatory synaptic transmission through activation of presynaptic A receptors. Amphetamine depresses neurotransmission through stimulation of astrocytes and the consequent A receptor activation. Furthermore, astrocytes modulate the acute behavioral psychomotor effects of amphetamine. Therefore, astrocytes mediate the dopamine- and amphetamine-induced synaptic regulation, revealing a novel cellular pathway in the brain reward system.Corkrum et al. report that astrocyte activity is required for dopamine- and amphetamine-evoked synaptic regulation and amphetamine-induced locomotor effects. Their study reveals astrocytes as active components of dopaminergic signaling and the brain reward system.

Charcot–Marie–Tooth disease (CMT) type 2A is a form of peripheral neuropathy, due almost exclusively to dominant mutations in the nuclear gene encoding the mitochondrial protein mitofusin-2 (MFN2). However, there is no understanding of the relationship of clinical phenotype to genotype. MFN2 has two functions: it promotes inter-mitochondrial fusion and mediates endoplasmic reticulum (ER)–mitochondrial tethering at mitochondria-associated ER membranes (MAM). MAM regulates a number of key cellular functions, including lipid and calcium homeostasis, and mitochondrial behavior. To date, no studies have been performed to address whether mutations in MFN2 in CMT2A patient cells affect MAM function, which might provide insight into pathogenesis. Using fibroblasts from three CMT2AMFN2 patients with different mutations in MFN2, we found that some, but not all, examined aspects of ER–mitochondrial connectivity and of MAM function were indeed altered, and correlated with disease severity. Notably, however, respiratory chain function in those cells was unimpaired. Our results suggest that CMT2AMFN2 is a MAM-related disorder but is not a respiratory chain-deficiency disease. The alterations in MAM function described here could also provide insight into the pathogenesis of other forms of CMT.

PARP-1 inhibition has recently been employed in both mono- and combination therapies in various malignancies including melanoma with both promising and contradicting results reported. Although deeper understanding of the underlying molecular mechanisms may help improving clinical modalities, the complex cellular effects of PARP inhibitors make disentangling of the mechanisms involved in combination therapies difficult. Here, we used two cytostatic agents used in melanoma therapies in combination with PARP inhibition to have an insight into cellular events using the B16F10 melanoma model. We found that, when used in combination with cisplatin or temozolomide, pharmacologic blockade of PARP-1 by PJ34 augmented the DNA-damaging and cytotoxic effects of both alkylating compounds. Interestingly, however, this synergism unfolds relatively slowly and is preceded by molecular events that are traditionally believed to support cell survival including the stabilization of mitochondrial membrane potential and morphology. Our data indicate that the PARP inhibitor PJ34 has, apparently, opposing effects on the mitochondrial structure and cell survival. While, initially, it stimulates mitochondrial fusion and hyperpolarization, hallmarks of mitochondrial protection, it enhances the cytotoxic effects of alkylating agents at later stages. These findings may contribute to the optimization of PARP inhibitor-based antineoplastic modalities.

It is unclear how the mitochondrial fusion protein Optic atrophy 1 (OPA1), which inhibits cristae remodeling, protects from mitochondrial dysfunction. Here we identify the mitochondrial F1Fo-ATP synthase as the effector of OPA1 in mitochondrial protection. In OPA1 overexpressing cells, the loss of proton electrochemical gradient caused by respiratory chain complex III inhibition is blunted and this protection is abolished by the ATP synthase inhibitor oligomycin. Mechanistically, OPA1 and ATP synthase can interact, but recombinant OPA1 fails to promote oligomerization of purified ATP synthase reconstituted in liposomes, suggesting that OPA1 favors ATP synthase oligomerization and reversal activity by modulating cristae shape. When ATP synthase oligomers are genetically destabilized by silencing the key dimerization subunit e, OPA1 is no longer able to preserve mitochondrial function and cell viability upon complex III inhibition. Thus, OPA1 protects mitochondria from respiratory chain inhibition by stabilizing cristae shape and favoring ATP synthase oligomerization.

Interneurons are critical for proper neural network function and can activate Ca2 signaling in astrocytes. However, the impact of the interneuron-astrocyte signaling into neuronal network operation remains unknown. Using the simplest hippocampal Astrocyte-Neuron network, i.e., GABAergic interneuron, pyramidal neuron, single CA3-CA1 glutamatergic synapse, and astrocytes, we found that interneuron-astrocyte signaling dynamically affected excitatory neurotransmission in an activity- and time-dependent manner, and determined the sign (inhibition vs potentiation) of the GABA-mediated effects. While synaptic inhibition was mediated by GABAA receptors, potentiation involved astrocyte GABAB receptors, astrocytic glutamate release, and presynaptic metabotropic glutamate receptors. Using conditional astrocyte-specific GABAB receptor (Gabbr1) knockout mice, we confirmed the glial source of the interneuron-induced potentiation, and demonstrated the involvement of astrocytes in hippocampal theta and gamma oscillations in vivo. Therefore, astrocytes decode interneuron activity and transform inhibitory into excitatory signals, contributing to the emergence of novel network properties resulting from the interneuron-astrocyte interplay.