3 studies of SPG4 mechanisms

Shedding light on treatment targets

Here are 3 studies investigating the mechanisms behind SPG4 HSP. They all focus on significant interactions between Spastin, the protein expressed by the SPAST gene, and proteins expressed by other genes.

Complicated SPG4 studied in mice. Mechanism of memory deficits and dementia found.

Spastin depletion in mice resulted in reduced synapse numbers, longer microtubules and impaired microtubule dynamics involving the motor (kinesin) proteins. When treated with healthy spastin, these deficits and impairments were corrected. These insights may be helpful in targeting future treatments in people with complicated SPG4.


Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown.

Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses.

Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

SOURCE: PLoS Biol. 2020 Aug 31;18(8):e3000820. doi: 10.1371/journal.pbio.3000820. eCollection 2020 Aug. PMID: 32866173

Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits

André T Lopes  1 Torben J Hausrat  1 Frank F Heisler  1 Kira V Gromova  1 Franco L Lombino  1 Timo Fischer  2 Laura Ruschkies  1 Petra Breiden  1 Edda Thies  1 Irm Hermans-Borgmeyer  3 Michaela Schweizer  4 Jürgen R Schwarz  1 Christian Lohr  2 Matthias Kneussel  1

  1. Department of Molecular Neurogenetics, Center for Molecular Neurobiology, ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
  2. Division of Neurophysiology, University of Hamburg, Hamburg, Germany.
  3. Transgenic Animal Unit, Center for Molecular Neurobiology, ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
  4. Morphology Unit, Center for Molecular Neurobiology, ZMNH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Spastin regulated by HIPK2 gene. Treatment restores levels to normal.

A protein expressed by the HIPK2 gene was found in this study to regulate Spastin dynamics in SPG4 mouse and differentiated human SPG4 neurons. Targeting the HIPK2/Spastin axis might be a strategy to develop Spastin-elevating therapeutic approaches.


Hereditary Spastic Paraplegia (HSP) is a neurodegenerative disease most commonly caused by autosomal dominant mutations in the SPG4 gene encoding the microtubule-severing protein spastin. We hypothesise that SPG4-HSP is attributable to reduced spastin function because of haploinsufficiency; thus, therapeutic approaches which elevate levels of the wild-type spastin allele may be an effective therapy. However, until now, how spastin levels are regulated is largely unknown.

Here, we show that the kinase HIPK2 regulates spastin protein levels in proliferating cells, in differentiated neurons and in vivo. Our work reveals that HIPK2-mediated phosphorylation of spastin at S268 inhibits spastin K48-poly-ubiquitination at K554 and prevents its neddylation-dependent proteasomal degradation. In a spastin RNAi neuronal cell model, overexpression of HIPK2, or inhibition of neddylation, restores spastin levels and rescues neurite defects.

Notably, we demonstrate that spastin levels can be restored pharmacologically by inhibiting its neddylation-mediated degradation in neurons derived from a spastin mouse model of HSP and in patient-derived cells, thus revealing novel therapeutic targets for the treatment of SPG4-HSP.

SOURCE: Life Sci Alliance. 2020 Oct 26;3(12):e202000799. doi: 10.26508/lsa.202000799. Print 2020 Dec. PMID: 33106322 © 2020 Sardina et al.

Spastin recovery in hereditary spastic paraplegia by preventing neddylation-dependent degradation

Francesca Sardina  1 Alessandra Pisciottani  2 Manuela Ferrara  2 Davide Valente  2   3 Marialuisa Casella  4 Marco Crescenzi  4 Angelo Peschiaroli  5 Carlo Casali  6 Silvia Soddu  3 Andrew J Grierson  7 Cinzia Rinaldo  8   3

  1. Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR), c/o Sapienza University, Rome, Italy
  2. Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR), c/o Sapienza University, Rome, Italy.
  3. Unit of Cellular Networks and Molecular Therapeutic Targets, IRCCS-Regina Elena National Cancer Institute, Rome, Italy.
  4. Core Facilities, Italian National Institute of Health, Rome, Italy.
  5. Institute of Translational Pharmacology, CNR, Rome, Italy.
  6. Department of Medico-Surgical Sciences and Biotechnologies, University of Rome Sapienza, Latina, Italy.
  7. Sheffield Institute for Translational Neuroscience, University of Sheffield, Sheffield, UK.
  8. Institute of Molecular Biology and Pathology (IBPM), National Research Council (CNR), c/o Sapienza University, Rome, Italy

Spastin loss alone not enough for defects to occur. Increased levels of Pak3 protein play a role.

A protein produced by glial cells*, Pak3, increases in spastin-deficient fruit fly larvae and is found necessary to cause neuronal defects. When both spastin and Pak3 are deficient, structural and nerve junction defects do not occur. There is the suggestion that a similar mechanism may underlie autosomal dominant HSPs in humans.

*Glial cells are non-neuronal cells in the brain, spinal cord and in the peripheral nervous system that do not produce electrical impulses. They maintain balance in the neuron and form myelin – the material that ensheathes neurons, providing support and protection.


Neurodegenerative mechanisms due to mutations in spastin currently center on neuronal defects, primarily in microtubule and endomembrane regulation. Spastin loss in Drosophila larvae compromises neuronal microtubule distribution, alters synaptic bouton morphology, and weakens synaptic transmission at glutamatergic neuromuscular junction (NMJ) synapses.

Pak3, a p21-activated kinase that promotes actin polymerization and filopodial projections, is required for these spastin mutant defects; animals lacking both genes have normal NMJs. Here we show that Pak3 is expressed in central and peripheral glial populations, and reduction of Pak3 specifically in subperineurial glial cells is sufficient to suppress the phenotypes associated with spastin loss. Subperineurial glia in the periphery ensheathe motor neuron axons and have been shown to extend actin-based projections that regulate synaptic terminals during normal NMJ development. We find that these subperineurial glial projections are Pak3-dependent and nearly twice as frequent in spastin mutants, while in Pak3, spastin double mutants, neither glial projections nor synaptic defects are observed.

Spastin deficiency thus increases Pak3-dependent subperineurial glia activity, which is in turn required for neuronal defects. Our results demonstrate a central role for Pak3-mediated, altered glial behavior in the neuronal defects due to spastin loss, and suggest that a similar reactive glia-mediated mechanism may underlie human AD-HSP pathogenesis.

SOURCE: Front Neurosci. 2020 Sep 4;14:912. doi: 10.3389/fnins.2020.00912. eCollection 2020. PMID: 33013303 Copyright © 2020 Ozdowski, Wentzell, Engert, Abbott and Sherwood.

Suppression of spastin Mutant Phenotypes by Pak3 Loss Implicates a Role for Reactive Glia in AD-HSP

Emily F Ozdowski  1 Jill S Wentzell  1 Stefanie M Engert  1 Helena Abbott  1 Nina T Sherwood  1

  1. Department of Biology, Duke University, Durham, NC, United States.

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