Figure 2. Morphologic changes occurring in diabetic axons. Diabetic axons present an increased number of microtubules and neurofilaments proximally, as well as increased cross-sectional area near the cell body. On the other hand, axonal cross-sectional area near the synapse is decreased, and the total number of microtubules and neurofilaments also decrease distally. As a consequence of cytoskeleton and nerve terminals derangements, motor proteins fail to bind microtubule end, contributing for the impairment of axonal transport.
Abnormal synthesis or post-translational modifications of the axonal cytoskeleton can not only influence axonal growth and caliber, but also impair nerve regeneration (Longo et al., 1986, Ekstrom and Tomlinson, 1989, Pekiner et al., 1996). Changes on retrograde signaling from the site of injury to the cell body were described in experimental diabetes (Fink et al., 1987, Logan and McLean, 1988), and the delivery of growth factors are also known to be affected (Jakobsen et al., 1981, Fernyhough et al., 1995).
It has been reported that experimental diabetes causes a reduction in the axonal transport of isotopically labelled unidentified proteins and glycoproteins in several types of peripheral nerves (Jakobsen and Sidenius, 1979, Sidenius and Jakobsen, 1979, Jakobsen and Sidenius, 1980, Sidenius and Jakobsen, 1980, Meiri and McLean, 1982). Studies regarding axonal transport rates in sciatic nerve of STZ-induced diabetic rats suggest that the primary event in the development of neurological abnormalities in diabetes is an impairment of the retrograde axonal transport (Jakobsen and Sidenius, 1979, Sidenius and Jakobsen, 1981), secondarily leading to the impairment of the anterograde transport of structural proteins (Jakobsen and Sidenius, 1980). Fast axonal transport is impaired at sciatic nerve and the changes found in protein and glycoconjugates synthesis and transport can be related to the early reduction in axon calibre and conduction velocity in peripheral nerve of STZ-induced diabetic rat (Sidenius and Jakobsen, 1979). Regarding retrograde axonal transport, diabetes also induces a decrease in peripheral axon organelle speed in sciatic nerve and dorsal and ventral nerve roots at 1 month of diabetes (Abbate et al., 1991). Moreover, axonal transport of receptors in the sciatic nerves of STZ-induced diabetic rats is affected, suggesting that impaired axonal transport of receptors may explain part of the neurological disturbances observed in diabetic patients (Laduron and Janssen, 1986). The reduction in fast transport rate in the sciatic nerve of diabetic rats was eliminated by maintenance of normal blood glucose levels with insulin administration (Meiri and McLean, 1982). Insulin treatment is able to prevent the slowing in transport occurring in diabetic rats and reverse an already slowed transport velocity (Sidenius and Jakobsen, 1982). In short-term experimental diabetes, defects in both anterograde and retrograde axonal transport of 6-phosphofructokinase activity were also detected, which were prevented by intensive insulin treatment (Willars et al., 1987). Moreover, decreased tubulin and actin transport rates are counteracted by ganglioside treatment suggesting a pharmacological effect that could be correlated with molecular interactions between integral membrane glycolipids and cytoskeletal elements (Figliomeni et al., 1992).
More recently, it was demonstrated that hyperglycemia impairs axonal transport in olfactory receptor neurons in mice. Increased oxidative stress in STZ-induced wild type diabetic mice activates the p38 MAPK pathway in association with phosphorylation of tau, attenuating axonal transport rates in the olfactory system. In STZ-induced superoxide dismutase-overexpressing mice, in which superoxide levels are reduced, these deficits are reversed (Sharma et al., 2010).
Diabetes also induces changes in the axonal transport of distal trophic factors to the soma. Increasing evidence shows that retrograde transport impairments of nerve growth factor (NGF) accounts for some functional deficits occurring in experimental diabetic neuropathy. Expression deficits in both NGF and its high-affinity receptor, trkA, were described in animal models of diabetes, leading to decreased retrograde axonal transport of NGF and decreased support of NGF-dependent sensory neurons, concomitant with decreased expression of neuropeptides expressed by these neurons, such as substance P and calcitonin gene-related peptide (Tomlinson et al., 1997). NGF and neurotrophin-3 (NT-3) retrograde transport was also found to be significantly reduced in the cervical vagus nerve in diabetic animals (Lee et al., 2001). In the sciatic nerve of STZ-induced diabetic rats, a clear reduction in the retrograde transport of NGF is detected (Hellweg et al., 1994). In STZ-induced diabetic rats the relative levels of phosphorylated (activated) c-Jun N-terminal kinase (JNK) and p38 retrogradly transported in sciatic nerve are increased compared with age-matched controls. Treatment of diabetic animals with NT-3 prevents the activation of JNK and p38, suggesting that JNK and p38 axonally transported mediate the transfer of diabetes-related stress signals, possibly triggered by loss of neurotrophic support, from the periphery to the sensory neuron soma (Middlemas et al., 2003). Moreover, it was also proposed that impaired PI3 kinase/Akt signal pathway may partly account for the reduced retrograde axonal transport of neurotrophins in the vagus nerve of STZ-induced diabetic rats (Cai and Helke, 2003).
Axonal transport impairments under diabetes in the CNS
Although most studies regarding changes in axonal transport triggered by diabetes have been focused in changes occurring at peripheral nervous system, evidences from studies in the optic nerve, spinal cord and brain also indicate that axonal transport is impaired at the central nervous system.
Analysis of slow axonal transport in the optic nerve of STZ-induced diabetic rats (4-6 weeks diabetes duration) and BioBreeding rats with spontaneous diabetes (2.5-3.5 months duration), showed impaired transport of NF subunits, tubulin and actin (Medori et al., 1985, Medori et al., 1988), further evidencing that cytoskeleton proteins transport is altered under diabetes. Moreover, a delay in the transport of 60, 52 and 30 kDa polypeptides, likely to be the glycolytic enzymes aldolase, neuron-specific enolase and pyruvate kinase, respectively, was also detected in both animal models of diabetes (Medori et al., 1988).
In the brain of STZ-induced diabetic rats, altered electrophoretic mobility of actin from a cytoskeletal protein preparation was detected, suggesting the presence of a product of nonenzymatic glycation. Furthermore, diabetes also leads to an increase in the phosphorylation of spinal cord neurofilament proteins. These post-translational modifications of neuronal cytoskeletal proteins in the spinal cords and brains of diabetic rats may contribute, as already referred, to the impairment of axonal transport and subsequent nerve dysfunction in experimental diabetes (McLean et al., 1992).
In the retina of diabetic rabbits, a reduction in slow axonal transport was observed, with a concomitant reduction in fast axonal transport and protein synthesis, whereas protein degradation remains unchanged (Chihara, 1981, Chihara et al., 1982). These evidences suggest that changes in axonal transport in the optic nerve may be a reflection of reduced protein synthesis in retinal ganglion cells (RGCs) (Tsukada and Chihara, 1986). Studies using fluoro-gold, a neuronal retrograde tracer, demonstrated a decreased accumulation of this tracer in RGCs of STZ-induced diabetic rats, thus suggesting that retrograde axonal transport from the dorsal lateral geniculate nucleus in the brain to the RGCs in the retina is impaired (Zhang et al., 1998, Zhang et al., 2000). Nevertheless, the accumulation of fluoro-gold in RGCs of OLETF rats, an animal model of type II diabetes with obesity, is not significantly decreased compared with control rats, suggesting that, the impairment of retrograde axonal transport in RGCs is higher in type 1 than in type 2 diabetic rats (Zhang et al., 1998). Changes in polyol pathway may play a role in the progressive impairment of retrograde axonal transport in the optic nerve of STZ-induced diabetic rats, suggesting that it may be a consequence of metabolic changes associated with diabetes (Ino-Ue et al., 2000). Besides changes in retrograde transport, more recently, it was found a deficit in the anterograde transport from the retina to the superior colliculus after induction of diabetes with STZ (Fernandez et al., 2012a, b).
In summary, potential changes in motor protein content and distribution previously described in the retina (Baptista et al., 2014), might underlie some changes already detected in axonal transport in the retina and visual pathway under diabetic conditions (Zhang et al., 2000, Fernandez et al., 2012a).
Mitochondria axonal transport deficits under diabetes
Since neurons are highly polarized cells, with long axons constituting a major challenge to the movement of proteins, vesicles, and organelles between cell bodies and presynaptic sites, axonal transport motor proteins require ATP demands, therefore implying the localization of functional mitochondria along the axons. The supply of appropriate levels of ATP is mainly produced by mitochondria via oxidative phosphorylation. The majority of mitochondria are produced in the cell body and transported along axonal microtubules by protein motors to reach areas with high ATP and calcium buffering requirements (Lin and Sheng, 2015). Distal cellular compartments such as synapses depend upon the efficient delivery of mitochondria through active transport to provide local sources with ATP. Generally, kinesin motors drive anterograde mitochondrial transport, while dyneins are responsible for retrograde transport. Nevertheless, single mitochondrion rarely move in only one direction (Lin and Sheng, 2015). Their transport along microtubules typically involves pauses of short and long duration and abrupt changes in direction, which suggests that individual mitochondrion are simultaneously coupled to kinesins, dyneins, and anchoring machinery whose actions compete or oppose one another (Figure 3). Figure 3. Axonal mitochondrial transport. In neurons, mitochondria can be observed to undergo dynamic, bidirectional transport along neuronal axons, changing direction frequently, pausing or switching to persistent docking. These complex mitochondrial mobility patterns are a result of mitochondrial coupling to anterograde and retrograde motor proteins.
Averaging the bidirectional and saltatory components yields a net mitochondrial velocity that falls between fast moving vesicles and slow-moving cytoskeletal proteins: 0.3-2.0 μm/s (Cai et al., 2011). Mobile mitochondria can become stationary or pause in regions that have a high metabolic demand and can move again rapidly in response to physiological changes. Defects in mitochondrial transport are implicated in the pathogenesis of several major neurological disorders (Sheng and Cai, 2012).
KIF5 motors are responsible for axonal transport of mitochondria. In KIF5A-/- neurons, the velocity of mitochondrial transport is reduced both in anterograde and retrograde direction (Karle et al., 2012). Decreased number of mitochondria in axons will likely decrease ATP supply to molecular motors leading to decreased anterograde and retrograde movement of both mitochondria and vesicles (De Vos et al., 2008). Growing evidence suggests that mitochondrial dysfunction play a significant role in neurodegenerative diseases like Huntington's disease, Alzheimer's disease and amyotrophic lateral sclerosis (Brownlees et al., 2002, Stamer et al., 2002, Zhao et al., 2010, Reddy, 2011).
It was already described that diabetes-induced nerve degeneration is mediated by alterations in mitochondrial ultrastructure and physiology (Fernyhough et al., 2010). Moreover, alterations in mitochondrial trafficking has also been proposed as a mediator of neurodegeneration in diabetic nerves (Fernyhough et al., 2010), but as far as we are concerned, there are no studies addressing the effect of diabetes on mitochondria axonal transport rates in the CNS. In the axons of hippocampal neurons exposed to elevated glucose, we did not detect any significant change in the intensity of fluorescence, distribution or number of accumulations related with mitochondria, when compared to control (Baptista et al., 2013). Nevertheless, we cannot exclude the possibility that mitochondria in the axons of hippocampal neurons are being affected by diabetes since other factors, besides hyperglycemia, may also have an impact in mitochondria transport. Neuroinflammation also plays a role in diabetes complications and may lead to changes in axonal transport of mitochondria. It was reported that tumor necrosis factor (TNF) induces perinuclear clustering of mitochondria by impairing kinesin-mediated transport in L929 cells (De Vos et al., 1998).
Neuroinflammation and axonal transport impairments
Neuroinflammation may contribute to changes in axonal transport (Figure 4). Pro-inflammatory cytokines, such as TNF and interleukin-1β, are upregulated in the hippocampus of diabetic BB/Wor rats (Sima et al., 2009) and STZ-induced diabetic animals (Kuhad et al., 2009). TNF receptor-1 activation induces the activation of kinase pathways, resulting in hyperphosphorylation of kinesin light chain and inhibition of kinesin activity, evidencing direct regulation of kinesin-mediated organelle transport by extracellular stimuli via cytokine receptor signaling pathways (De Vos et al., 2000). Exposure of hippocampal neuronal cultures to TNF enhances the phosphorylation of JNK in neurites. TNF treatment induces the dissociation of KIF5B from tubulin in axons and inhibits axonal transport of mitochondria and synaptophysin by reducing the mobile fraction via JNK (Stagi et al., 2006). Nitric oxide released from activated microglia, inhibits axonal movement of synaptic vesicle precursors containing synaptophysin and synaptotagmin in hippocampal neurons, suggesting that disturbance of axonal transport by microglia-derived nitric oxide may therefore be responsible for axonal injury and synaptic dysfunction in brain diseases characterized by neuroinflammation (Stagi et al., 2005). Moreover, hydrogen peroxide, a common reactive oxygen species elevated during inflammation, also inhibits axonal transport in hippocampal cultures (Fang et al., 2012) (Figure 4). Further studies will be needed to determine if similar pathways may be active under diabetic conditions, therefore contributing for previously detected changes in axonal transport.
Figure 4. Microglia-driven inflammation and axonal transport impairment. In hippocampal cultures, nitric oxide (NO) released from activated microglia inhibits axonal movement of synaptic vesicles. Tumor necrosis factor (TNF) produced by activated glial cells in inflammatory or degenerative neurological diseases affects neurites by acting on the kinesin-tubulin complex and inhibiting axonal mitochondria and synaptophysin transport in hippocampal cultures. Moreover, hydrogen peroxide (H2O2), a common reactive oxygen species elevated during inflammation, also inhibits axonal transport.
Conclusions
Defects in axonal transport are early pathogenic events that may contribute for the development and progression of diabetic complications such as diabetic neuropathy, retinopathy and encephalopathy. Disruption of axonal transport can occur through several ways, including alterations in the cytoskeleton, molecular motors, cargoes (via inhibition of their attachment to motors), mitochondrial damage, and through microglia-mediated release of inflammatory mediators (via inhibition of motor attachment to microtubules). Nevertheless, little is known regarding the cellular and molecular mechanisms associated with axonal transport impairments under diabetes. Advances in the understanding of such mechanisms, may be crucial to develop novel neuroprotective strategies for diabetic complications.