The neuroprotective effects observed in this study demonstrate the feasibility and efficacy of focal transplantation-based astrocyte replacement for ALS and also show that targeted multi-segmental cell delivery to cervical spinal cord is a promising therapeutic strategy, particularly because of it relevance to addressing respiratory compromise associated with ALS. Over the last 10 years, accumulating data has implicated non-neuronal cells in the pathogenesis of neuronal degeneration in ALS. Starting from the earliest observation of functional abnormalities in ALS astrocytes18,28
to the more recent studies in chimeric mice8
, the aberrant function of cells, including microglia7
, that surround motor neuron somas and dendrites appears to contribute to disease progression. Chimeric mutant SOD1 expression models suggest that increasing the proportion of healthy wild-type non-neuronal cells is inversely related to measures of disease severity such as animal survival8
, similar to the GRP transplantation effects presented in the current study. In these chimeric animals, the presence of wild-type non-neuronal cells (likely astrocytes and microglia) in the spatial vicinity of mutant SOD1 expressing motor neurons prevents pathological changes in these neurons. More recent studies demonstrate that the reduction of mutant SOD1 selectively from astrocytes using LoxSOD1G37R
mice results in a prolongation of disease duration, but has no effects on disease onset13
. These results suggest a particular role for astrocytes in later progression of disease. In aggregate, these studies suggest that replacement of abnormal astroglia - or enrichment of healthy astroglia - with normal functioning precursors, could be one approach towards focally altering the microenvironment around motor neurons.
Dysfunction of astrocyte glutamate transport, specifically GLT1 (EAAT2), is found in humans with ALS and in animal models of ALS6,18
, and may be a contributing factor in disease progression. Loss of this astroglial protein is known to cause excitotoxic motor neuron degeneration15
. Furthermore, ALS astrocytes alter the expression of motor neuron dendritic glutamate (AMPA) receptors, also making them more susceptible to excitotoxicity. Small molecule drug screening for agents that enhance glial glutamate transporter function shows that increasing astroglial GLT1 can be beneficial in ALS models29
. More recently, several in vitro studies of immature ALS rodent astrocytes also suggest abnormal properties resulting in motor neuron cell death9,11
, although the exact mechanisms remain unknown. Thus, any approach to offset or replace altered astroglial function - especially in mature astrocytes - may be of therapeutic benefit.
In the current work, GRP transplants were able to partially prevent loss of total tissue GLT1 levels in cervical cord, thereby targeting one important, and ALS-relevant, function of astrocytes. The demonstration that GLT1−/−
GRPs did not have any effects on behavioral measures or animal survival also suggests that glutamate-relevant pathways may contribute to the cascade of events leading to cell death in this model and that the focal beneficial effects of GRP transplantation could be explained, at least in part, by increases in glutamate transporter expression. While disease duration is extended by reducing mutant SOD1 from astrocytes in the LoxSOD1G37R
mice, GLT1 loss in lumbar spinal cord sections is not dependent on the presence of mutant SOD1 in astrocytes13
. GLT1 loss may instead be related to non-cell autonomous damage to astrocytes from SOD1 synthesis by other cells. Alternatively, alterations in neuron-astrocyte communication, as a result of SOD1-mediated neuronal injury, could be responsible. Several possible explanations could account for the discrepancy between the current observations and the previous study13
. These include the anatomical location of tissue sampling (both cervical and lumbar spinal cord versus lumbar only), time frame of sampling (at a specific time point during disease course versus end-stage), as well as the contributions of GLT1 loss to death in different species carrying different SOD1 mutations that result in differences in disease course itself (less than 180 days in the SOD1G93A
rat model versus greater that 375 days in the SOD1G37R
mouse model). Finally, it is also possible that the increases in GLT1 that we observed in our model may represent only one of several related pathways relevant to astrocytic influences on disease progression. The lack of further increases in behavioral and neuroprotective measures from the transplantation of GLT1-overexpressing GRPs may be related to the observation that increases in GLT1 levels were relatively small compared to previous in vitro studies24
and in vivo drug studies29
Astrogliosis with GFAP up-regulation is a central feature of ALS and SOD1 pathology, and previous studies have noted that some protective molecules down-regulate GFAP expression in ALS models30
. We did not observe any detrimental effects on disease by the introduction of GFAP+
GRP-derived astrocytes. These results suggest that astrogliosis may not only be reflected by increases in GFAP expression, but also by other astrocyte factors which may influence disease progression. Furthermore, the direct effect of astrogliosis itself is unclear as reactive astrocytes also play important protective roles in other CNS injury paradigms31
Neural precursor cell transplantation offers a strategy for slowing neurodegenerative disease progression and/or promoting recovery of function because engrafted cells have the potential of replacing lost or dysfunctional neurons and glia. Previous neural precursor transplantation studies in motor neuronopathies have focused mostly on motor neuron replacement32–35
; however, this is a challenging strategy for neurodegenerative diseases because of problems associated with motor neuron differentiation, establishment of appropriate circuitry with host neurons, and extension to and connectivity with musculature.
Other transplantation strategies not based on motor neuron replacement, including those with enhanced trophic factor production, also show promise in ALS models36–47
. When transplanted into the lumbar spinal cord of SOD1G93A
rats, human cortical NPCs that over-express the trophic factor, GDNF, provide some, albeit limited, neuroprotection48,49
. These transplants provide a neuroprotective effect on motor neuron survival, but do not promote efficacy with respect to improved hind-limb motor performance and animal survival, possibly due to lack of astrocyte differentiation in vivo. Our study did not suggest that there was a pattern of significant increases in VEGF, IGF-1 or BDNF neurotrophic factor secretion to account for the observed pathological or behavioral phenotypes. However, it is possible that at the cellular level some neurotrophic factor secretion may have played a role in the efficacy of GRPs, but was not appreciated in the whole tissue analysis of the cervical spinal cord in this study.
These results serve as a proof-of-principle that stem cell transplantation-based astrocyte replacement is feasible and a potentially viable option for ALS therapy. Delivery to the cervical spinal cord targets key motor neuron pools which ultimately affect survival in ALS patients, and respiratory measures remain the most reliable for use in ALS clinical trials.
Glial precursors are particularly promising candidates for astrocyte replacement because of their robust survival, efficient astrocytic differentiation, and lack of tumor formation. While more immature cell classes such as multipotent neural stem cells and pluripotent embryonic stem cells are desirable sources for transplant derivation, the current work suggests that the use of more mature lineage-restricted progenitors may be an optimal strategy for achieving targeted phenotypic replacement.