Sonic Hedgehog Controls the Phenotypic Fate and Therapeutic Efficacy of Grafted Neural Precursor Cells in a Model of Nigrostriatal Neurodegeneration.

The expression of soluble growth and survival promoting factors by neural precursor cells (NPCs) is suggested to be a prominent mechanism underlying the protective and regenerative effects of these cells after transplantation. Nevertheless, how and to what extent specific NPC-expressed factors contribute to therapeutic effects is not well understood. Using RNA silencing, the current study investigated the roles of two donor NPC molecules, namely glial cell-line derived neurotrophic factor (GDNF) and sonic hedgehog (SHH), in the protection of substantia nigra dopamine neurons in rats treated with 6-hydroxydopamine (6-OHDA). Analyses indicate that as opposed to the knock-down of GDNF, SHH inhibition caused a profound decline in nigrostriatal neuroprotection. Further, SHH silencing also curbed endogenous neurogenesis and the migration of host brdU+/dcx+ neural precursors into the striatum, which was present in the animals receiving control or GDNF silenced NPCs. A change in graft phenotype, mainly reflected by a reduced proportion of undifferentiated nestin+ cells, as well as a significantly greater host microglial activity, suggested an important role for these processes in the attenuation of neuroprotection and neurogenesis upon SHH silencing. Overall these studies reveal core mechanisms fundamental to grafted NPC-based therapeutic effects, and delineate the particular contributions of two graft-expressed molecules, SHH and GDNF, in mediating midbrain dopamine neuron protection, and host plasticity after NPC transplantation

High-Frequency Stimulation of the Rat Entopeduncular Nucleus Does Not Provide Functional or Morphological Neuroprotection from 6-Hydroxydopamine

Deep brain stimulation (DBS) is the most common neurosurgical treatment for Parkinson\u27s disease (PD). Whereas the globus pallidus interna (GPi) has been less commonly targeted than the subthalamic nucleus (STN), a recent clinical trial suggests that GPi DBS may provide better outcomes for patients with psychiatric comorbidities. Several laboratories have demonstrated that DBS of the STN provides neuroprotection of substantia nigra pars compacta (SNpc) dopamine neurons in preclinical neurotoxin models of PD and increases brain-derived neurotrophic factor (BDNF). However, whether DBS of the entopeduncular nucleus (EP), the homologous structure to the GPi in the rat, has similar neuroprotective potential in preclinical models has not been investigated. We investigated the impact of EP DBS on forelimb use asymmetry and SNpc degeneration induced by 6-hydroxydopamine (6-OHDA) and on BDNF levels. EP DBS in male rats received unilateral, intrastriatal 6-OHDA and ACTIVE or INACTIVE stimulation continuously for two weeks. Outcome measures included quantification of contralateral forelimb use, stereological assessment of SNpc neurons and BDNF levels. EP DBS 1) did not ameliorate forelimb impairments induced by 6-OHDA, 2) did not provide neuroprotection for SNpc neurons and 3) did not significantly increase BDNF levels in any of the structures examined. These results are in sharp contrast to the functional improvement, neuroprotection and BDNF-enhancing effects of STN DBS under identical experimental parameters in the rat. The lack of functional response to EP DBS suggests that stimulation of the rat EP may not represent an accurate model of clinical GPi stimulation

Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain

Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson\u27s disease. Although viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be because of the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing Parkinson\u27s disease and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared with the young adult. We examined recombinant adeno-associated virus 2/5-mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra aged rats displayed 40%-60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Furthermore, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP messenger RNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less messenger RNA and protein expression

Effect of SHH and GDNF silencing on the number of grafted NPCs.

<p>We assessed the number of GFP expressing donor cells (A) in both the striatum and nigra of rats receiving control and silenced NPCs. Estimates of grafted GFP⁺ cells in hematoxylin counterstained tissue sections (B) showed no significant differences in the striatum or SN of SHH or GDNF silenced animals when compared to controls. However the animals receiving GDNF+SHH silenced grafts showed reduced numbers of grafted cells in the striatum in comparison to animals with control cells (C). Values are expressed as mean ± SEM [*p<0.05, **p<0.01, ***p<0.001 compared to shC, two way ANOVA with Bonferroni’s post test].Scale bars: A—25 μm; B—10 μm.</p

SHH knockdown increases microglial activity in the host brain.

<p>Host microglia expressing cd11b and cd68 antigens (expressed by activated microglia) were analyzed two weeks after NPC implantation. A greater number of both cd68⁺ and cd11b⁺ cells were noted in animals that had received the SHH or GDNF+SHH silenced NPCs (D-F, J-L, P-R, V-X high mag images in b, d, f, g) in comparison to rats with control (A-C, M-O; high mag images in a and e) or GDNF silenced (G-I, S-U; high mag images in c and g) grafts. The number of cd11b⁺ and cd68⁺ cells present in regions adjacent to the transplanted NPCs were quantified using confocal microscopy, and it was confirmed that the decrement of SHH in grafted NPCs had in fact resulted in a significant (p<0.001) activation of microglia in the host neural environment (Y, Z). Values are expressed as mean ± SEM [***p<0.001 compared to control, one way ANOVA with Bonferroni’s post test]. Scale bar: A-S—50 μm, a-f—15 μm.</p

Inhibition of SHH influences the phenotypic fate of grafted NPCs.

<p>We analyzed the co-expression of antigens specific to nestin (undifferentiated NPCs, A-C), immature neurons (tuj1, D-F), astrocytes [(s100β (G-I) and oligodendroglia [RIP (J-L)] in the GFP NPCs. Counts of grafted GFP cells indicated that while the number of nestin⁺ cells was significantly lower, compared to control NPCs in donor cells silenced for SHH or GDNF and SHH, shGDNF grafts did not show such an effect (a). This was true for grafts both in the striatum as well as SN. Representative images of nestin stained grafts are shown in M, Q, U. The GDNF silenced grafts on the other hand showed a significant (p<0.001) reduction in the number of cells expressing the neuron marker tuj1 (b). Such a decline in tuj1 expression was not observed in the SHH silenced NPCs. Representative images of tuj1 stained grafts are shown in O, S, W. Further, the number of S100β⁺ cells was greater in striatal grafts with SHH knock-down, with no such influence observed in any of the other experimental groups (c). Representative images of S100β⁺ cells in grafts are shown in N, R, V. Finally, with respect to oligodendrocyte differentiation, no significant differences were observed between control and silenced NPCs (d). Representative images of RIP staining in grafts are shown in P, T, X. Values are expressed as mean ± SEM [*p<0.05, **p<0.01, ***p<0.001 compared to shC, one way ANOVA with Bonferroni’s post test]. Scale bars: A-L—10 μm; M-X—30 μm.</p

<i>In vitro</i> lentiviral silencing of GDNF and SHH in NPCs.

<p>Western blotting analyses of cultured NPCs indicated that the cells expressed only GDNF (~25kda) and SHH (~45kda), but no detectable SDF1α (~11kda), under basal conditions (E-G; GDNF and SHH were run on the same gel and membrane divided, whereas SDF1 was run on a separate gel). Therefore, an FIV based <i>in vitro</i> RNA interference approach was used to knock down the expression of GDNF, SHH or both in donor NPCs before transplantation (B). A schematic diagram of pVETL construct used for expression of shRNAs and the GFP reporter is depicted in (A). After testing, the two most efficient shRNA constructs targeting GDNF or SHH were cloned into a FIV eGFP vector and subsequently infected into the NPCs in culture (D, shows a representative image of <i>in vitro</i> NPCs infected with control eGFP vector). The NPCs did not show a significant change in survival and proliferative ability after the shRNA infections (H, I). When the silencing efficiency of the virally infected shRNA’s was examined through mRNA (qPCR; J, K) and protein (western blot; L-O) analyses, it was determined that in particular the GDNF1 and SHH4 constructs were able to most significantly (p<0.001) inhibit GDNF and SHH expression respectively, when compared to mock (eGFP vector) and scrambled (non-targeted shRNA) controls, and were focused upon in the rest of the study. Image E displays control FIV eGFP expressing NPCs <i>in vivo</i>, 2 weeks after transplantation. Values are expressed as mean ± SEM [*p<0.05, **p<0.01, ***p<0.001 compared to scrambled control, one-way ANOVA with Tukey’s post hoc test]. Scale bars: A, B, C (in C)– 25 μm; D, E—100 μm.</p