Evidence for prodromal changes in neuronal excitability and neuroinflammation in the CA3 region of the hippocampus in young alpha-synuclein (A30P) transgenic mice and investigation of the effect of intranasal metformin treatment

Abstract

The toxic aggregation of the pre-synaptic protein alpha (α)-synuclein is a key feature of several neurodegenerative diseases, including Lewy body dementias (LBDs). Patients with LBDs have progressive cognitive impairment including memory loss and complex visual hallucinations. Recent evidence from patient and rodent models has shown neuronal hyperexcitability and neuroinflammation in most neurodegenerative diseases. Novel research on metformin, a type 2 diabetes drug, has demonstrated anti-inflammatory/neuroprotective properties and can easily penetrate the blood-brain barrier. I investigated hyperexcitability and neuroinflammatory changes in the hippocampus of young hA30P mice, a transgenic mouse model that over-expresses human mutant alpha-synuclein and tested the effects of metformin treatment on these changes. Immunofluorescence staining was conducted using sections of the hippocampus from 1-4 months hA30P mice and wild-type (WT) mice as a control. Primary antibodies for neuronal, glial, and neuroinflammatory markers were employed. Proteome cytokine array and label-free mass spectrometry (LF-MS) measurements were made from hippocampal tissue extracts. I also employed intranasal metformin treatment in WT and hA30P mice, followed by immunofluorescence histology. Immunofluorescence revealed significant differences between WT and hA30P animals in the CA3 region of the hippocampus. An interesting biphasic change in the expression of c-Fos in male hA30P mice was observed with high expression at 1 month, consistent with early onset of hyperexcitability, but lower expression from 2-4months in male hA30P mice compared to the controls, possibly indicating chronic hyperexcitability. Neuroinflammation was indicated by significant increases in the % area of GFAP, a marker for reactive astrocytes, and the number of Iba-1+ microglia that expressed iNOS immunoreactivity in 1-4 months male hA30P mice compared to WT. A similar increase in %GFAP was observed in female hA30P mice, however, the %Iba-1 area was not different between female WT and hA30P mice. In WT mice aged 2-4 months only 4.6% of Iba-1+ cells co-expressed iNOS. In contrast, in age-matched hA30P mice, 87% of cells co-expressed Iba-1 and iNOS. There was a significant increase in GFAP and Iba-1/iNOS co-expression immunoreactivity in a cohort of 1- month-old hA30P compared to WT mice. A significant decrease in NeuN positive neurons and perineuronal nets detected with WFA lectin was also observed in the CA3 region of hA30P mice compared to WT at 2-4 months. The results of LF-MS at 2 months in the hippocampus of male hA30P mice revealed that α-synucleinopathy causes significant changes in the proteome, indicating the effect of overexpressed human mutant α-synuclein. There were 158 significantly upregulated proteins in the hippocampus of hA30P mice involved in neurotransmission, and neuronal activity, such as Tenm1, Wdfy3, Syn, and Lin7, mitochondrial dysfunction such as TOMM6 and Rala, oxidative stress such as Rab 1A, and apoptosis in neurons such as Snrk. At the same time, there were 30 significantly downregulated proteins involved in different biological function e.g. DNA/RNA damage such as Gmpr2, Rps7, and Rbmx. Although there was no direct evidence for neuroinflammation through proteins associated with microglial and astrocytic activation, there were many proteins related to inflammation such as Ddrgk1, Cadps, Syn2, Hspa1l, Hspa2, Hspa5, Hspa8, Lin7a, Lin7b, and Lin7c that showed upregulation in hA30P compared to WT mice. In a pilot experiment, metformin intranasal treatment significantly reduced the proportion of activated microglia in the CA3 hippocampal region in treated versus vehicle hA30P mice, however, other neuronal and glial markers showed no significant differences. Overall, my data showed evidence for early network hyperexcitability and neuroinflammation that might contribute to the progression of neurodegenerative changes. Further experiments are needed to explore the therapeutic potential of metformin.