Elsevier

Neurobiology of Disease

Volume 47, Issue 2, August 2012, Pages 174-183
Neurobiology of Disease

AAV2 mediated retrograde transduction of corticospinal motor neurons reveals initial and selective apical dendrite degeneration in ALS

https://doi.org/10.1016/j.nbd.2012.03.036Get rights and content

Abstract

Corticospinal motor neurons (CSMN) are the cortical component of motor neuron circuitry, which controls voluntary movement and degenerates in diseases such as amyotrophic lateral sclerosis, primary lateral sclerosis and hereditary spastic paraplegia. By using dual labeling combined with molecular marker analysis, we identified AAV2–2 mediated retrograde transduction as an effective approach to selectively target CSMN without affecting other neuron populations both in wild-type and hSOD1G93A transgenic ALS mice. This approach reveals very precise details of cytoarchitectural defects within vulnerable neurons in vivo. We report that CSMN vulnerability is marked by selective degeneration of apical dendrites especially in layer II/III of the hSOD1G93A mouse motor cortex, where cortical input to CSMN function is vastly modulated. While our findings confirm the presence of astrogliosis and microglia activation, they do not lend support to their direct role for the initiation of CSMN vulnerability. This study enables development of targeted gene replacement strategies to CSMN in the cerebral cortex, and reveals CSMN cortical modulation defects as a potential cause of neuronal vulnerability in ALS.

Highlights

► Retrograde transduction approach is developed to selectively target CSMN in the motor cortex. ► AAV2–2 is effective for CSMN retrograde transduction in WT and hSOD1G93A mice. ► Cytoarchitecture of vulnerable CSMN is revealed with very high precision and detail. ► CSMN degeneration starts from the apical dendrite as they lose spines and connections in layer II/III. ► The presence of astrogliosis and microglia activation do not lend support for their direct role in CSMN vulnerability.

Introduction

In amyotrophic lateral sclerosis (ALS), unlike other motor neuron diseases, both the cortical and spinal components of motor neuron circuitry progressively degenerate (Brown and Robberecht, 2001, Ravits et al., 2007). Therefore, bringing effective treatments to ALS requires targeting both the spinal motor neurons (SMN) in the spinal cord and the corticospinal motor neurons (CSMN) in the motor cortex. CSMN degeneration results in lack of cortical input and impairs voluntary movement in other motor neuron disorders, such as primary lateral sclerosis and hereditary spastic paraplegia (Fink, 2002, Fink, 2006, Rainier et al., 2003). Therefore, due to the central involvement of CSMN in motor neuron circuitry and their selective vulnerability and degeneration in various diseases, it is imperative to understand cellular mechanisms for CSMN vulnerability and to develop CSMN directed gene delivery approaches in the cerebral cortex.

Viral vectors offer a great tool for gene therapy and replacement studies in CNS (Kootstra and Verma, 2003, Verma and Weitzman, 2005). Recombinant, adeno-associated viruses (AAV) are ideal candidates for retrograde transduction of projection neurons and are extensively used to target SMN. AAV2-IGF-1 injected into the muscle of hSOD1G93A ALS transgenic mice, one of the best-characterized mouse models of ALS (Gurney et al., 1994), retrogradely transduced SMN and increased life span (Kaspar et al., 2003). Intravascular administration of AAV9 (Foust et al., 2009) and intrathecal injections of AAV6 and AAV9 at the level of the lumbar spinal cord transduced SMN throughout the spinal cord (Snyder et al., 2011). Intramuscular delivery of AAV6 encoding silencer shSOD1 RNA transduced SMN, but failed to alter disease course in the hSOD1G93A mouse (Towne et al., 2011). Moreover, CST fibers were labeled and mixed neuron populations were transduced by direct AAV injection into the motor cortex (Hutson et al., 2011), but effective retrograde transduction of CSMN has never been achieved, even though AAV-mediated gene therapy approaches have been considered for ALS (Nizzardo et al., 2011).

In an effort to build gene delivery approaches selectively targeting CSMN and to reveal cytoarchitectural changes that correlate with cellular mechanisms underlying CSMN vulnerability, we used an AAV-mediated retrograde transduction strategy coupled with cellular analysis. We investigated whether a distinct set of AAV serotypes can be used to retrogradely transduce CSMN following precise injection into the corticospinal tract (CST), without affecting other spinal cord neurons and circuitries. AAV2–2 was found to be most effective in CSMN gene delivery in both wild-type (WT) and hSOD1G93A transgenic ALS mice, which show early and selective CSMN degeneration (Ozdinler et al., 2011). Retrograde transduction of CSMN with an enhanced green fluorescent protein (eGFP) gene fully revealed the cytoarchitecture of CSMN at a level of detail not previously possible by other retrograde labeling approaches (Hains et al., 2003, Ozdinler et al., 2011, Shokouhi et al., 2010).

We report spine loss, as well as severe and selective apical dendrite degeneration, especially in layer II/III of motor cortex in the hSOD1G93A transgenic ALS mice at P60, whereas basal dendrites are unaffected. Cellular interaction studies of CSMN with astrocytes and microglia, cell types that are extensively studied with respect to disease initiation and progression (Alexianu et al., 2001, Hall et al., 1998, Levine et al., 1999), revealed close cell–cell interactions especially with degenerating hSOD1G93A CSMN both at the soma and apical dendrite. Our results set the stage for future targeted gene delivery approaches to CSMN and validate the use of hSOD1G93A mouse for further gene therapy approaches for ALS. In addition, our findings suggest that an intrinsic cellular defect affecting distal apical dendrites, especially in layer II/III of motor cortex, underlies connectivity and modulation deficiencies of CSMN as a potential mechanism for their vulnerability and degeneration.

Section snippets

Mice

All procedures were approved by the Northwestern University Animal Care and Use Committee and conformed to the standards of the National Institutes of Health. WT and hSOD1G93A transgenic ALS mice, in C57BL/6 background, were obtained from Jackson laboratories (Gurney et al., 1994). Genotypes of mice were determined as described by the vendor.

Adeno-associated virus (AAV) serotype generation

AAV vectors with different serotypes were generated by the University of Pennsylvania Vector Core facility by triple transfection of subconfluent HEK293

AAV-mediated retrograde transduction of CSMN

CSMN can be retrogradely labeled by red fluorescent microspheres or FluoroGold injections into CST at the C2–C3 level of the spinal cord in the postnatal mice (Ozdinler and Macklis, 2006, Ozdinler et al., 2011). Using this approach, we studied the possibility of targeted gene delivery to CSMN using AAV and determined the AAV serotype with the highest transduction efficiency in both WT and hSOD1G93A mice. CST injections performed using matched-titers of AAV2–1, AAV2–2, AAV2–5, AAV2–6, AAV2–7,

Discussion

There are two main limitations in bringing effective cellular therapies to neurodegenerative diseases; one is our lack of understanding for the cellular basis of selective neuronal vulnerability, and the second is the absence of gene delivery approaches targeting only vulnerable neurons within the complex structure of the cerebral cortex. In this study, we overcome both of these limitations. By developing and applying a novel AAV-mediated retrograde transduction approach, which transduces CSMN

Acknowledgments

This work has been supported by grants from the Les Turner ALS Foundation, Wenske Foundation, Brain Research Foundation (P.H.O), Northwestern University Translational Innovation grant (P.H.O. and M.C.B). J.H.J. was supported by the ALSA Safenowitz postdoctoral fellowship, and S.R.V. by the NIH T32-GM08061 Pre-doctoral training grant. We would like to thank, J. Xie for excellent technical assistance and W. Weber for help with experiments. We would also like to thank B. Goosens from the

References (60)

  • M. Santello et al.

    TNFalpha controls glutamatergic gliotransmission in the hippocampal dentate gyrus

    Neuron

    (2011)
  • S.W. Scheff et al.

    Quantitative assessment of cortical synaptic density in Alzheimer's disease

    Neurobiol. Aging

    (1990)
  • C. Towne et al.

    Neuroprotection by gene therapy targeting mutant SOD1 in individual pools of motor neurons does not translate into therapeutic benefit in fALS mice

    Mol. Ther.

    (2011)
  • G. Zanette et al.

    Different mechanisms contribute to motor cortex hyperexcitability in amyotrophic lateral sclerosis

    Clin. Neurophysiol.

    (2002)
  • M.E. Alexianu et al.

    Immune reactivity in a mouse model of familial ALS correlates with disease progression

    Neurology

    (2001)
  • B. Alstermark et al.

    In vivo recordings of bulbospinal excitation in adult mouse forelimb motoneurons

    J. Neurophysiol.

    (2004)
  • C.T. Anderson et al.

    Sublayer-specific microcircuits of corticospinal and corticostriatal neurons in motor cortex

    Nat. Neurosci.

    (2010)
  • S. Boillee et al.

    Onset and progression in inherited ALS determined by motor neurons and microglia

    Science

    (2006)
  • R.H. Brown et al.

    Amyotrophic lateral sclerosis: pathogenesis

    Semin. Neurol.

    (2001)
  • L.I. Bruijn et al.

    Unraveling the mechanisms involved in motor neuron degeneration in ALS

    Annu. Rev. Neurosci.

    (2004)
  • S. Daya et al.

    Gene therapy using adeno-associated virus vectors

    Clin. Microbiol. Rev.

    (2008)
  • S.T. DeKosky et al.

    Synapse loss in frontal cortex biopsies in Alzheimer's disease: correlation with cognitive severity

    Ann. Neurol.

    (1990)
  • J.L. Eberling et al.

    Functional effects of AAV2-GDNF on the dopaminergic nigrostriatal pathway in parkinsonian rhesus monkeys

    Hum. Gene Ther.

    (2009)
  • J.K. Fink

    Hereditary spastic paraplegia: the pace quickens

    Ann. Neurol.

    (2002)
  • J.K. Fink

    Hereditary spastic paraplegia

    Curr. Neurol. Neurosci. Rep.

    (2006)
  • K.D. Foust et al.

    Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons

    Hum. Gene Ther.

    (2008)
  • K.D. Foust et al.

    Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes

    Nat. Biotechnol.

    (2009)
  • G. Gowing et al.

    Ablation of proliferating microglia does not affect motor neuron degeneration in amyotrophic lateral sclerosis caused by mutant superoxide dismutase

    J. Neurosci.

    (2008)
  • J.M. Graham et al.

    Diffusion tensor imaging for the assessment of upper motor neuron integrity in ALS

    Neurology

    (2004)
  • J. Grosskreutz et al.

    Widespread sensorimotor and frontal cortical atrophy in Amyotrophic Lateral Sclerosis

    BMC Neurol.

    (2006)
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